Regulation of Mmp2/9 Transport to the Invadopodia

REGULATION OF MMP2/9 TRANSPORT TO THE INVADOPODIA

DURING BREAST CANCER CELL INVASION

ABITHA JACOB

B.S., Monash University, 2005

M.S., University of Dundee, 2007

A thesis submitted to the

Faculty of the Graduate School of the

University of Colorado in partial fulfillment

of the requirements for the degree of

Doctor of Philosophy

Molecular Biology Program

2016

This thesis for the Doctor of Philosophy degree by

Abitha Jacob

has been approved for the

Molecular Biology Program

Andrew Bradford, Chair

Heide Ford

Paul Jedlicka

Mary Reyland

Traci Lyons

Rytis Prekeris, Advisor

Date 05-20-2016

Jacob, Abitha (Ph.D., Molecular Biology)

Regulation of MMP2/9 Transport to the Invadopodia During Breast Cancer Cell Invasion

Thesis directed by Professor Rytis Prekeris

ABSTRACT

Invadopodia-dependent degradation of the basement membrane plays a major role during metastasis of breast cancer cells. Basement membrane degradation is mediated by targeted secretion of various matrix metalloproteinases (MMPs). Specifically, MMP2 and

MMP9 possess the ability to hydrolyze components of the basement membrane and were shown to regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates MMP2/9 targeting to the invadopodia during cancer cell invasion remains to be defined. Rab GTPases are master regulators of intracellular vesicle transport. Here, we identify and characterize the role of Rab40b GTPase as a protein required for secretion of MMP2/9. Using a combination of shRNA based protein knockdown in

MDA-MB-231 breast cancer cells with fluorescent microscopy and in situ zymography, we report that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. We identify Rab40b as novel mediator for the targeting of MMP2/9 secretory vesicles to the forming invadopodia and is required for invadopodia-dependent extracellular matrix degradation. Furthermore, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. Using a 3D invasion and spheroid culture system, we show that the results we obtained from the 2D system is relevant in a 3D system that simulates the in vivo environment better.

Following our analysis of Rab40b in MDA-MB-231 cells, we sought to determine whether Rab40b regulates breast cancer cell invasion in vivo. Utilizing shRNA based stable

iii knockdown cell lines and a mouse mammary fat pad injection model, we demonstrate the role of Rab40b in primary tumor growth and metastasis. We show that Rab40b depletion results in reduced primary tumor size, possibly due to decreased angiogenesis and also lack invasion and cell dispersal. Additionally, we show decreased micro-metastases in the mouse lung upon depletion of Rab40b. Taken together, our data suggest that Rab40b decreases tumor growth and metastasis, potentially as a direct result of cancer cell invasion as well as effects on primary tumor angiogenesis.

We also demonstrate that Rab40b functions by interacting with Tks5, a known Src kinase substrate and invadopodia-regulating protein. Significantly, the expression of Rab40b is increased in highly metastatic basal breast tumors, and we demonstrate that Rab40b and

Tks5 levels are regulated by miR-204. We show that miR-204 is involved in down-regulating

Rab40b and Tks5, thus inhibiting MMP2/9 targeting to invadopodia resulting in a decrease in invadopodia-associated ECM degradation. This is the first study that identifies and defines a novel Rab40b/Tks5 and miR-204 dependent invadopodia transport pathway that regulates

MMP2/9 secretion and extracellular matrix remodeling during breast cancer progression both in vitro and in vivo.

The form and content of this abstract are approved. I recommend its publication.

Approved: Rytis Prekeris

TABLE OF CONTENTS

CHAPTER

I. THE ROLE OF MMPS AND INVADOPODIA DURING CANCER CELL INVASION AND METASTASIS ...... 1

Abstract ...... 1

The role of invadopodia in cancer cell invasion ...... 2

Stages of invadopodia formation ...... 4

Initiation ...... 4

Assembly...... 5

Maturation ...... 6

Invadopodia formation and function in vivo ...... 7

The role of matrix metalloproteinases in cancer cell metastasis ...... 8

Mechanisms mediating MMP targeting to invadopodia ...... 11

Targeting of MMP14 ...... 12

Targeting of MMP2 and MMP9 ...... 15

Conclusions and future objectives ...... 16

II. RAB40B REGULATES MMP2 AND 9 TARGETING TO THE INVADOPODIA DURING BREAST CANCER CELL INVASION ...... 17

Abstract ...... 17

Introduction ...... 18

Materials and methods ...... 20

Antibodies and constructs ...... 20

Cell culture and tet-inducible MDA-MB-231 lines...... 21

siRNA library screen...... 21

Elisa assays ...... 22

In vitro invasion and motility assays ...... 22

Fluorescent microscopy ...... 23

FACS analysis ...... 24

Zymography assays ...... 25

In situ zymography assays ...... 25

Reverse transcriptase PCR and quantitative PCR ...... 26

Flow cytometry based GFP-hGH secretion assay ...... 27

Statistical analyses ...... 28

Results ...... 28

Rab40b GTPase is required for MMP2 and MMP9 secretion ...... 28

Rab40b is required MMP2 and MMP9 sorting and secretion ...... 33

Rab40b is localized to VAMP4 containing secretory vesicles ...... 36

Rab40b is required for breast cancer cell invasion and invadopodia dependent ECM degradation in vitro ...... 41

Discussion ...... 47

III. THE ROLE AND REGULATION OF RAB40B/TKS5 COMPLEX IN MMP TARGETING AND CANCER CELL INVASION ...... 52

Abstract ...... 52

Introduction ...... 53

Materials and Methods ...... 55

Antibodies and constructs ...... 55

Cell lines ...... 56

GST-Rab40b affinity chromatography ...... 56

GST-Tks5 fragments and glutathione bead pull-down assay ...... 57

Molecular modeling ...... 57

Inverse inversion assay ...... 58

In situ zymography ...... 58

RT-PCR and qPCR ...... 59

Luciferase assay ...... 60

3D spheroid assay ...... 60

Mouse mammary fat pad xenograft assays ...... 61

In situ analysis of lung metastasis ...... 62

Immunohistochemistry ...... 62

Breast cancer data mining ...... 63

Results……………………………………………………………………63

Rab40b is required for breast cancer cell invasion and invadopodia extension ...... 63

Rab40b is required for primary tumor growth and metastasis in vivo ...... 73

Tks5 is a Rab40b binding protein that regulates invadopodia function ...... 79

miR-204 regulates expression of Rab40b and Tks5 expression breast cancer cells ...... 83

Discussion ...... 88

IV. CONCLUSIONS AND FUTURE DIRECTIONS...... 95

Conclusions ...... 95

Future directions ...... 96

REFERENCES ...... ……………………………………………………………………104

vii

LIST OF FIGURES

Figure

1.1 The schematic representation of the pathways regulating MMP14 and MMP2/9 targeting to the invadopodia ...... 13

2.1 Characterization of MDA-MB-231 cell lines expressing tet-inducible MMP2-myc or MMP9-myc ...... 30

2.2 VAMP4 is present on MMP2/9 transport organelles ...... 31

2.3 MMP2 and MMP9 secretion regulating proteins identified from siRNA screen ...... 32

2.4 Rab40b knockdown decreases MMP2 and MMP9 secretion in MDA-MB-231 cells ...... 34

2.5 Rab40b increases lysosomal degradation of MMP2 and MMP9...... 35

2.6 FLAG-Rab40b co-localizes with VAMP4-containing secretory vesicles ...... 37

2.7 VAMP4 is required for MMP2/9 secretion ...... 39

2.8 Localization of Rab40bcontaining organelles during cell invasion in vitro ...... 40

2.9 MMP2-myc and MMP9-myc accumulate within the invadopodia during cell invasion ...... 42

2.10 Rab40b is required for MDA-MB-231 cell invasion in vitro ...... 43

2.11 Rab40b is required for invadopodia-associated ECM degradation ...... 45

2.12 MMP2/9 are required for invadopodia-associated ECM degradation...... 46

2.13 Rab40b is required for invadopodia-associated ECM degradation in HMCB cells...... 48

3.1 Quantification of Rab40b in Rab40b-KD cell lines ...... 65

3.2 Rab40b localizes to the invadopodia and regulates cell invasion in 3D ...... 66

viii

3.3 Rab40b is required for invadopodia extension and maturation...... 68

3.4 Rab40b affects the ability of spheroids to form invasive strands…...... 70

3.5 Rab40b knockdown affects angiogenesis and primary tumor growth and metastasis in vivo...... 71

3.6 Rab40b depletion affects survival and metastases in mice ...... 72

3.7 Proliferation and apoptosis in tumors from mice terminated at 8 weeks ...73

3.8 Proliferation and apoptosis in tumors from mice terminated at 2cm3 tumor burden...... 75

3.9 Tks5 is a Rab40b binding partner ...... 77

3.10 Rab40b binds to the PX domain of Tks5 ...... 79

3.11 Rab40b mRNA levels in different grades and subtypes of breast tumors..82

3.12 MiR-204 regulates Rab40b ...... 83

3.13 MiR-204 seed regions in Tks5 and effects of miR-204 on Rab40b and Tks5 in other breast cancer lines ...... 85

3.14 MiR-204 regulates Tks5 ...... 87

3.15 Model for Rab40b mediated MMP2/9 targeting to the invadopodia...... 92

3.16 Model for Rab40b mediated Src degradation and invadopodia formation through actin regulation ...... …..99

CHAPTER I

THE ROLE OF MMPS AND INVADOPODIA DURING CANCER

CELL INVASION AND METASTASIS 1

Abstract

The dissemination of cancer cells from the primary tumor to a distant site, known as metastasis, is the main cause of mortality in cancer patients. Metastasis is a very complex cellular process that involves many steps, including breaching of the basement membrane to allow the movement of cells through tissues. The basement membrane breach occurs via highly regulated and localized remodeling of the extracellular matrix (ECM), which is mediated by formation of structures, known as invadopodia. Recently, invadopodia have emerged as key cellular structures that regulate the metastasis of many cancers. Invadopodia are plasma membrane protrusions that the cells utilize to break through the basement membrane. Furthermore, targeting of various cytoskeletal modulators and matrix metalloproteinases (MMPs) has been shown to play a major role in regulating invadopodia function. This chapter focuses on the different stages of invadopodia formation and function during cancer cell invasion and the role that MMPs play in this process. The current proposed mechanisms and machinery utilized for targeting MMPs to the invadopodia are described, with an emphasis on MMPs known to be enriched at the invadopodia.

1This chapter of the thesis is largely based on our previously published review Jacob,A., and Prekeris, R. 2015. The regulation of MMP targeting to invadopodia during cancer metastasis. Frontiers In Cell and Developmental Biology. 3(4): 1-9.

The Role of Invadopodia in Cancer Cell Invasion

Metastasis is a very complex process that involves the dissemination of cancer cells from the primary tumor to a distant secondary site. Cells need to traverse the basement barrier to metastasize. While the mechanisms mediating the movement of cells through the basement membrane remain to be fully characterized, it is now widely accepted that the formation of actin rich invasive protrusions is a key step during cancer cell invasion. These structures were identified in tissue culture cells and have been termed podosomes or invadopodia (Chen, 1989; Tarone et al., 1985). While the functional differences between podosomes and invadopodia remain to be clearly defined, recent nomenclature has tried to distinguish podosomes as present in normal cells and invadopodia as present in cancer cells

(Hoshino et al., 2013; Murphy and Courtneidge, 2011b). Nevertheless, there are more similarities than differences between podosomes and invadopodia. Both these structures are actin rich and possess the ability to degrade the ECM (Linder and Kopp, 2005). However, they differ in their size, number, lifetime and location, which allows for differentiation between them during visualization (Linder and Kopp, 2005). Both podosomes and invadopodia are usually visualized with phalloidin, which stains filamentous actin and appears as punctate spots located below the nucleus. Podosomes have been observed in cells of monocytic lineage such as macrophages (Lehto et al., 1982; Linder et al., 1999) osteoclasts (Marchisio et al., 1984) and in induced smooth muscle cells (Gimona et al., 2003) as well as endothelial cells (Moreau et al., 2003; Osiak et al., 2005). In contrast, invadopodia are found in cells transformed with oncogenes (David-Pfeuty and Singer, 1980; Tarone et al.,

1985) and are thought to protrude further into the matrix and invade more aggressively than podosomes (Linder et al., 2011; Murphy and Courtneidge, 2011b; Weaver, 2008b). A variety

2 of actin regulators, scaffold proteins, small GTPases and proteinases have been shown to play important roles in several steps of invadopodia formation. Several studies using animal xenografts and primary tumor cells from patients have also demonstrated the formation of invadopodia in vivo. Additionally, invadopodia have been observed in bladder cancer (Sutoh et al., 2010; Yamamoto et al., 2011), colorectal cancer (Schoumacher et al., 2010), breast cancer (Coopman et al., 1998; Yamaguchi et al., 2005a; Yamaguchi et al., 2011) squamous cell carcinoma (Takkunen et al., 2010) and glioblastoma (Stylli et al., 2008).

The ability of invadopodia to degrade ECM is attributed to the presence of matrix degrading enzymes such as matrix metalloproteinases (MMPs). While the cellular machinery mediating the targeted release of MMPs from invadopodia remains to be defined, it is becoming clear that some

MMPs are targeted and released from invadopodia to facilitate invasion (Nakahara et al., 1997).

MMP14, MMP2 and MMP9 have all been shown to be important in cancer progression and enriched at the invadopodia (Artym et al., 2006; Bourguignon et al., 1998; Clark and Weaver, 2008; Monsky et al.,

1993; Nakahara et al., 1997; Poincloux et al., 2009a). MMP2 and MMP9 contain fibronectin repeats that help them recognize gelatin (denatured collagen) as a substrate (Polette et al.,

2004) and Type IV collagen is the main constituent of the BM, one of the first barriers that cancer cells need to traverse to metastasize. In addition, MMP14 can recognize and cleave a broad spectrum of ECM substrates and also functions as an activator of MMP2 (Lebeau et al., 1999). Thus, the combined activity of MMP2, MMP9 and MMP14 is suggested to be an important step in initiating localized degradation of the BM during epithelial cancer metastasis (Chen and Wang, 1999; Nakahara et al., 1997). Even though this chapter discusses only the proteolytic aspect of MMP14, it is interesting to note that it can also function through a non-proteolytic mechanism. MMP14 can stimulate ATP production by activating

Hypoxia- Inducible Factor-1(HIF-1) (Sakamoto and Seiki, 2010). The non-proteolytic activity of MMP14 also includes binding of its transmembrane domain to integrin β1, which leads to MAPK activation, thereby regulating branching in mammary epithelium (Mori et al.,

2013).

Stages of Invadopodia Formation

Invadopodia formation and function are complex cellular events that involve substantial reorganization in cytoskeleton dynamics and membrane transport. Recent studies have attempted to define different stages of invadopodia formation and function by using various criteria, including the recruitment of actin, targeted release of MMPs and the localized degradation of the ECM. Based on these, invadopodia formation has been divided into three stages, namely, initiation, assembly and maturation. The following sections describe the stages of invadopodia formation in detail.

Initiation

In the initiation phase of invadopodia formation, invadopodial precursors or ‘buds’ form at the cell periphery which are usually marked by actin puncta (Yamaguchi et al.,

2005b). The process of invadopodia formation is initiated by growth factors such as epidermal growth factor (EGF), platelet derived growth factor (PDGF) and transforming growth factor- β (TGFβ). Growth factor receptor signaling activates Phosphatidylinositide 3-

Kinase (P13K) leading to Src activation, which in turn phosphorylates multiple proteins including Tks5 (Tyrosine kinase substrate). Since the PX domain (phospholipid binding domain) of Tks5 has been shown to bind to PI3P and PI(3,4)P2 (Abram et al., 2003), it was

suggested that Tks5 localizes to PI(3,4)P2 enriched regions of the plasma membrane, thus targeting Tks5 to initiate the invadopodia ‘bud’ (Courtneidge et al., 2005b). Src

4 phosphorylates synaptojanin 2 to activate its phosphatase activity, which dephosphorylates

PI(3,4,5)P3 at the plasma membrane to form PI(3,4)P2, thus forming the site for invadopodia formation (Chuang et al., 2012). Src mediated activation of the Abl-family kinase Arg also leads to the phosphorylation of cortactin, resulting in the recruitment of Nck1 to the invadopodia (Mader et al., 2011; Oser et al., 2009). Nck1 then recruits the Neural Wiskott-

Aldrich syndrome protein (N-Wasp) complex to the invadopodia leading to Cdc42-dependent activation of N-Wasp. N-Wasp in turn induces actin polymerization through the Arp2/3 complex, resulting in formation of invadopodial precursors (Yamaguchi et al., 2005a). The co-localization of cortactin with Tks5 in invadopodial ‘buds’, led to the hypothesis that Tks5 acts a scaffolding protein that recruits the other cellular components required for initiation of invadopodia formation. However, some recent evidence suggests that Tks5 might instead be involved in invadopodia maturation (Sharma et al., 2013).

Assembly

Invadopodia are highly dynamic and motile structures that have been divided into two types based on their motility and lifetimes, motile short-lived and stationary long-lived invadopodia (Yamaguchi et al., 2005a). The short-lived invadopodia are thought to be precursors of fully functional mature invadopodia and could also be equivalent to podosomes

(Yamaguchi et al., 2005a). In vitro, newly formed or early invadopodia can move laterally within the plasma membrane that faces the ECM. These motile invadopodia are then anchored and stabilized by actin polymerization and extension of the invadopodia

(Yamaguchi et al., 2005a). A plethora of proteins are recruited to the invadopodial ‘bud’ converting it from motile to stationary invadopodia. The precise order of how proteins are recruited is still unknown. Since Tks5 has five tandem SH3 domains, it is thought that Tks5

5 can scaffold several proteins like Nck1, Nck2, Grb2 (Growth factor receptor bound protein 2) and N-Wasp. It has also been proposed that Tks5, along with cortactin, recruits various actin regulators leading to nucleation of branched actin filaments and the formation of a stable actin core in the invadopodia (Clark and Weaver, 2008; Oser et al., 2009). Consistent with this hypothesis, it has been shown that phosphorylation of cortactin leads to dissociation of the cortactin/cofilin complex, which is an essential step in invadopodia formation and elongation (Oser et al., 2009). The dissociation of the cortactin/cofilin complex and the polymerization of actin is also a pH-dependent process (Frantz et al., 2008; Pope et al.,

2004).

Maturation

Actin polymerization is crucial for formation and function of invadopodia. Inhibition of cofilin results in the formation of short-lived unstable invadopodia, which suggests that actin polymerization caused by cofilin is required for elongation and maturation of invadopodia (Yamaguchi et al., 2005a). Apart from the branched actin network, invadopodia also contain linear actin bundles (Li et al., 2010; Schoumacher et al., 2010). mDia2, a formin that induces the formation of linear actin networks, has been found to promote elongation and stability of invadopodia (Lizarraga et al., 2009). Fascin, another actin bundling protein has also been shown to promote elongation, stability and matrix degradation in invadopodia

(Li et al., 2010; Schoumacher et al., 2010).

Src kinase is a major regulator of invadopodia formation and function. Interestingly, several other protein kinases including Abl kinases like Arg (Abl related gene) kinase have recently emerged as important players in invadopodia formation and maturation (Beaty et al.,

2013). It was shown that β1 integrin interacts with Arg leading to stimulation of Arg

6 mediated cortactin phosphorylation, a key switch in promoting invadopodial maturation

(Beaty et al., 2013). Additionally, β1 integrin has been shown to localize to invadopodia and promote degradation of collagen type IV, the main constituent of the basement membrane

(Sameni et al., 2008), presumably by recruiting and docking proteases at the invadopodia.

Separase, a gelatinolytic enzyme that has been shown to be enriched at the invadopodia, binds to α3β1 integrin resulting in the formation of functional invadopodia (Mueller et al.,

1999). The ultrastructure of mature invadopodia has shown the presence of microtubules and many vesicles/endosomes indicative of active trafficking and a possible route for delivery of specific proteins like MMPs (Schoumacher et al., 2010). The activity of proteases docked at the invadopodia has been shown to be pH-dependent (Greco et al., 2014). The acidification of the peri-invadopodial space by the Na+/H+ exchanger (NHE1) promotes ECM proteolysis

(Busco et al., 2010).

Invadopodia Formation and Function In Vivo

Although there is an increasing amount of evidence demonstrating the existence of invadopodia in vitro, the formation and function of invadopodia in vivo is less well understood due to challenges associated with visualizing and distinguishing these structures in animal models. Cancer invasion usually occurs deep in tissues and these events are highly dynamic and unpredictable making it difficult to visualize invadopodia during primary tumor metastasis. Though the majority of invadopodial studies have been conducted in 2D tissue culture systems, some groups have studied invadopodia formation in 3D matrices as they better simulate the physiological in vivo environment. Such studies of invadopodia in complex 3D matrices have shown that the matrix degrading activity is localized to the base rather than the tip of the invadopodia (Tolde et al., 2010; Wolf et al., 2007). These 3D studies

7 have also helped establish criteria for the identification of invadopodia in vivo and provide a good model to study formation of invadopodia.

Despite the challenges mentioned above, there is some compelling evidence drawn from elegant in vivo experiments that confirm that invadopodia are not just in vitro artifacts.

Recently, the chorioallantoic membrane of the chicken embryo was used to visualize the intravascular formation of invadopodia and the extravasation of tumor cells into the stroma

(Leong et al., 2014). The same group also showed that knocking down invadopodial components like cortactin, Tks4 and Tks5 decreases extravasation of cells into the lung stroma in tail vein injected mice. Intravital live animal imaging has also allowed the visualization of MtLn3-GFP (a highly invasive rat mammary carcinoma line) invading into blood vessels using protrusions identified as invadopodia-like structures using invadopodia markers such as cortactin and N-WASP (Lohmer et al., 2014). Using live-cell imaging, it was shown that during the uterine-vulval development in Caenorhabditis elegans, the anchor cell breaches the uterine and vulval basement membranes by making an invadopodium

(Hagedorn et al., 2013). The Src-Tks5 pathway was shown to be required for the migration of neural crest cells using actin-rich protrusions in zebrafish (Murphy et al., 2011). Finally, it has also been shown that the intestinal epithelium of the zebrafish mutant meltdown forms invadopodia-like protrusions that invade into the stromal tissue in response to cues from the surrounding smooth muscle layer (Seiler et al., 2012).

The Role of Matrix Metalloproteinases in Cancer Cell Metastasis

Although epithelial cancers are one of the leading causes of death, the mechanisms regulating the development and metastasis of carcinomas are not fully understood. Multiple studies suggest that the progression of tumors is dependent on the intrinsic properties of cancer cells, such as their ability to

8 migrate and invade. Furthermore, many extrinsic factors, such as extracellular matrix (ECM) proteins, are also crucial for regulation of cancer metastasis. The ECM proteins that make up the specialized basement membrane (BM) serve as a barrier for cell invasion. However, the basement membrane which is rich in laminin and collagen IV, also provides the substrate for adhesion of the migrating tumor cells. Furthermore, BM degradation results in the release/activation of various growth factors required for angiogenesis, tumor growth and metastasis (Kalluri, 2003; Yurchenco, 2011). ECM degrading enzymes known as matrix metalloproteinases (MMPs) are known to play important roles in the degradation of the BM. Since several excellent reviews have already described the importance of

MMPs in cancer cell growth and metastasis (Deryugina and Quigley, 2006; Egeblad and Werb, 2002b;

Fingleton, 2006; Gialeli et al., 2011), this chapter will focus on the mechanisms governing the targeting of MMPs to invadopodia.

BM disruption involves a localized degradation of the ECM via the secretion of MMPs

(Chamber and Matrisian, 1997). MMPs are a family of zinc endopeptidases that cleave ECM molecules and are subdivided into categories depending on their substrate specificity. The MMP family of enzymes includes not only the classical secreted and membrane bound MMPs, but also ADAMs (a disintegrin and metalloproteinase). ADAM metalloproteinases, also known as sheddases, are involved in cleaving various growth factors, cytokines, receptors and adhesion molecules and are fundamental to development and homeostasis (Edwards et al., 2008). Like ADAMs, MMPs are also required for normal processes like tissue remodeling in embryonic development, wound healing, involution of mammary glands, angiogenesis and ossification (Woessner, 1991). However, high levels of MMPs or aberrant MMP expression have often been correlated with pathological conditions like periodontitis, arthritis (Woessner, 1991) and have been implicated in multiple stages of cancer progression including invasion and metastasis (Egeblad and Werb, 2002b). This chapter focuses on the canonical MMPs,

9 more specifically the ones that are targeted to the invadopodia and implicated in BM remodeling during metastasis of epithelial cancers. The association of MMPs with malignancies has been well documented with the majority of the evidence derived from mouse model studies and analysis of human patient samples. Based on substrate recognition, MMPs are categorized into interstitial collagenases, gelatinases, stromelysins and membrane bound MMPs. Out of the 28 known MMPs, 14 have been implicated in cancer development and progression (Kohrmann et al., 2009). It has been shown that elevated expression of MMP1, 2, 3, 7, 9, 13 and 14 is positively correlated with tumor progression, metastasis and poor overall prognosis (Ala-aho and Kahari, 2005; Bjorklund and Koivunen, 2005;

Hofmann et al., 2005; Kerkela and Saarialho-Kere, 2003; Lochter et al., 1998; Mook et al., 2004;

Wagenaar-Miller et al., 2004). Recently, it was shown that MMP9 drives tumor progression and metastasis of triple negative breast cancer (Mehner et al., 2014) and increased expression of MMP9 has been found in the early steps of melanoma (van den Oord et al., 1997). Expression of MMP9 has been associated both positively and negatively with survival rates in breast and colon cancer patients

(Pacheco et al., 1998; Scorilas et al., 2001; Takeha et al., 1997; Zeng et al., 1996). Additionally, cancer cells have lesser capability to colonize the lungs of MMP2 or MMP9 deficient mice compared to wild type mice (Itoh et al., 1999; Itoh et al., 1998a) and cancer cell proliferation is decreased in tumors obtained from MMP9 knock-out mice (Bergers et al., 2000; Coussens et al., 2000). Overexpression of

MMP3 and MMP14 (also known as MT1-MMP) has been shown to promote mammary carcinogenesis (Ha et al., 2001; Sternlicht et al., 1999). MMP12 expression in colon cancer has been correlated with increased survival (Yang et al., 2001). These studies indicate that several MMPs play a key role in cancer growth and metastasis. However, the expression levels and functions of individual

MMPs are clearly dependent on the stage and type of cancer.

While MMP expression is increased in many cancers, the levels of activated rather than total MMPs appear to be a better indicator of tumor metastatic potential. There are two main mechanisms of post transcriptional regulation of MMP activity: activation of the latent precursor form (zymogen) and inhibition of the active enzyme by tissue inhibitors of matrix metalloproteinases or TIMPs (Polette et al., 2004). Most MMPs are secreted in an inactive pro-enzyme form and are activated extra-cellularly. An interesting property of MMPs is that they are capable of mutual activation. For example, MMP1 and MMP14 can activate MMP2

(Murphy and Crabbe, 1995; Sang et al., 1996; Strongin et al., 1995). The proteolytic activity of MMPs is also regulated by tissue inhibitors of metalloproteinases (TIMPs), by binding to the zinc ion in their catalytic site (Fernandez-Catalan et al., 1998; Gomis-Ruth et al., 1997).

There are four known TIMPs, of which TIMP1 and TIMP2 are the most promiscuous and inhibit the majority of MMPs. In vivo studies in mice have shown that overexpression of

TIMP1 decreases metastasis to the brain and to the liver (Kruger et al., 1997; Kruger et al.,

1998; Soloway et al., 1996; Sternlicht and Werb, 2001). Overall, MMP activity is tightly regulated by different mechanisms and is involved in both normal and pathologic processes

(Polette et al., 2004).

Mechanisms Mediating MMP Targeting to Invadopodia

The final maturation stage of the invadopodia involves targeted delivery and exocytosis of MMP2, MMP9 and MMP14. The appearance of these MMPs is generally considered to be a mark of functional mature invadopodia. As a result, much effort has been invested in understanding the regulation of MMP targeting to invadopodia, leading to recent studies defining the machinery governing MMP transport during cancer cell invasion.

Targeting of MMP14

MMP14 is a membrane embedded MMP whose extracellular proteolytic activity is regulated by a balance between exocytosis and internalization via clathrin and/or caveolar mediated endocytosis (Remacle et al., 2003) (Figure 1.1A). Once internalized, MMP14 is then either targeted to lysosomes for degradation (Jiang et al., 2001; Remacle et al., 2003) or shunted to endocytic recycling pathways, thus controlling the levels of active enzyme at the cell surface (Remacle et al., 2003) (Figure 1.1A). However, invasive cancer cells have mechanisms to counteract the removal of the active enzyme from the plasma membrane.

Consistently, enrichment of active MMP14 at the invadopodia associated plasma membrane of tumor cells has been demonstrated in vitro (Artym et al., 2006; Clark and Weaver, 2008;

Nakahara et al., 1997; Steffen et al., 2008). Endocytic recycling (Poincloux et al., 2009a) exocytosis (Monteiro et al., 2013), Rab 8 (Bravo-Cordero et al., 2007) and Tks4 (Buschman et al., 2009) have all been shown to be involved in the localization of MMP14 to the invadopodia. However, the exact mechanism governing MMP14 targeting remains to be fully understood. Rab8 GTPase has been shown to be involved in mobilization of MMP14 from intracellular storage compartments allowing polarized recruitment of MMP14 to the invasive front of cells (Bravo-Cordero et al., 2007). Some of the exocytic components shown to regulate delivery of MMP14 to the invadopodia are cortactin, the Exocyst complex (consists of 8 subunits) and VAMP7 (vesicle associated membrane protein 7). Cortactin has been shown to regulate the cell surface expression of MMP14 (Clark et al., 2007). Sec8, a subunit

Figure 1.1. The schematic representation of the pathways regulating (A) MMP14 and (B) MMP2/9 targeting to the invadopodia.

13 of the Exocyst complex, was shown to localize at the invadopodia and is required for

MMP14 targeting to the invadopodia (Monteiro et al., 2013) (Figure 1.1A). Active RhoA and

Cdc42 trigger the interaction between the Exocyst subunits Sec3 and Sec8 and the polarity protein IQGAP1. This interaction has been shown to be required for the accumulation of

MMP14 at the invadopodia (Sakurai-Yageta et al., 2008). The Exocyst complex has also been shown to interact with Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) to ensure focal delivery of MMP14 to the invadopodia (Monteiro et al.,

2013). Since exocytosis depends on SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) which drive the fusion of transport vesicles with the plasma membrane, several recent studies have investigated the role of SNAREs in mediating

MMP14 transport. Consequently, it was shown that VAMP7 is required for trafficking of

MMP14 to invadopodia (Steffen et al., 2008) (Figure 1.1A).

Transport vesicle targeting and fusion with its destination membranes often relies on specific tethering factors that impart specificity to membrane transport. The tethering factors regulating MMP14 targeting remain to be identified. However, Tks4, a scaffolding factor related to Tks5, has been shown to be required for the formation and function of invadopodia

(Buschman et al., 2009). In the absence of Tks4, recruitment of MMP14 to podosomes is inhibited, implicating the role of Tks4 in targeting of MMP14 to the invadopodia (Buschman et al., 2009) (Figure 1.1A). Additionally, cortactin was reported to have a novel role in invadopodial maturation and invasion by regulating secretion of MMP14 at the invadopodia

(Clark and Weaver, 2008). However, it remains to be tested whether Tks4 and cortactin function as MMP14-vesicle tethers, or whether they affect MMP14 targeting indirectly by regulating the actin cytoskeleton within during invadopodia formation and maturation.

Recently, it was also shown that Orai1 calcium channel mediated Ca2+ oscillations regulate the proteolytic activity of invadopodia by allowing more MMP14 to be recycled to the plasma membrane (Sun et al., 2014).

Targeting of MMP2 and MMP9

MMP2 and MMP9 are gelatinases that possess fibronectin type II repeats that allow them to degrade collagen and gelatin. Gelatinolytic degradation can cause the release of signaling molecules from the ECM which aid cell migration and angiogenesis. A lot of effort has been focused on understanding the transport and targeting of gelatinases because they are overexpressed in a variety of tumors and are associated with tumor aggressiveness and poor patient prognosis (Egeblad and Werb, 2002b; Hiratsuka et al., 2002; Pacheco et al., 1998; van 't Veer et al., 2002). Although MMP2 and MMP9 have been shown to be enriched at the invadopodia, there is not much known about how these proteases are transported and targeted to the invadopodia. It has been reported that MMP2 and MMP9 are stored and transported in small vesicles that move along microtubules powered by kinesin in human melanoma cells

(Schnaeker et al., 2004) (Figure 1B). Similar to MMP14, the secretion of MMP2 and MMP9 were also shown to depend on cortactin (Clark and Weaver, 2008). Interestingly, in contrast to MMP14, endocytic transport and the Exocyst complex do not appear to play a role in targeted transport of MMP2 and MMP9 (Jacob et al., 2013). Much of the machinery mediating the transport of MMP2 and MMP9 has never been described and remains to be defined. This thesis will describe in detail with both in vitro and in vivo evidence, a novel network of machinery involved in the targeting of MMP2/9 to the invadopodia to facilitate cancer cell invasion and metastasis.

Conclusions and Future Objectives

Significant advances have been made in understanding the formation and function of invadopodia. However, there are still a lot more unknowns regarding this subcellular structure. While all of the above mentioned studies have helped to confirm the physiological role of invadopodia as a structure used by invasive cells to penetrate the basement membrane, more evidence is required to elucidate the functional role of invadopodia in vivo and understand how widespread the use of invadopodia by cells is. Many questions regarding the importance of invadopodia in cancer invasion and metastasis still exist. Future studies in the field of invadopodia will need to focus on detection of invadopodia in human cancer samples as well as to identify the role of invadopodia in the different steps of the metastatic cascade. The other areas that require focus are the identification of components specific to invadopodia that can be targeted and the development of compounds that can specifically inhibit invadopodia formation and function. These issues will need to be addressed before invadopodia can become a candidate for development of new cancer therapies.

The other interesting feature about invadopodia is that they are enriched in matrix degrading enzymes, MMP14, MMP2 and MMP9 being a few. There are well established pathways describing MMP14 transport. However, there is very little or almost nothing known about how MMP2 and MMP9 get transported and targeted to the invadopodia. The following chapters will discuss a novel pathway involved in transporting MMP2/9 during cancer cell invasion and metastasis and how these MMPs are important in regulating invadopodia formation and function.

CHAPTER II

RAB40B REGULATES MMP2 AND MMP9 TARGETING TO THE

INVADOPODIA DURING BREAST CANCER CELL INVASION 2

Abstract

MMP9 possess the ability to hydrolyze components of the basement membrane and were shown to regulate various aspects of tumor growth and metastasis. However, the membrane transport machinery that mediates MMP2/9 targeting to the invadopodia during cancer cell invasion remains to be defined. Since Rab GTPases are key regulators of membrane transport, we screened a human Rab siRNA library and identified Rab40b GTPase as a protein required for secretion of MMP2/9. We also have shown that Rab40b functions during at least two distinct steps of MMP2/9 transport. First, we demonstrate that Rab40b is required for MMP2/9 sorting into VAMP4-containing secretory vesicles. Second, we show that

Rab40b mediates the targeting of MMP2/9 secretory vesicles to the forming invadopodia and is required for invadopodia-dependent extracellular matrix degradation. Finally, we demonstrate that Rab40b is also required for breast cancer cell invasion in vitro. Based on these findings, we propose that Rab40b mediates MMP2/9 sorting and targeting to the forming invadopodia during breast cancer cell metastasis.

2This chapter of the thesis is largely based on our previously published article Jacob,A., Jing, J., Lee, J., Schedin,P., Gilbert, S.M., Peden, A., Junutula, A.R. and Prekeris, R. 2013.Rab40b regulates MMP2 and MMP9 targeting to the invadopodia during breast cancer cell invasion. Journal of Cell Science. 126: 4647-4658.

Introduction

The basement membrane is made up of a network of extracellular matrix (ECM) proteins, which serves as a barrier for cell motility and invasion. Loss of this barrier function is an important step in cancer cell invasion and metastasis (Roskelley and Bissell, 1995; Roskelley et al., 1995).

Furthermore, ECM degradation can lead to increased adhesion of the migrating cells and also result in the release and/or activation of various growth factors required for angiogenesis, tumor growth and metastasis (Roskelley et al., 1995). Basement membrane disruption usually involves a localized degradation of the ECM via targeted secretion of matrix metalloproteinases (MMPs) (Polette et al.,

2004; Visse and Nagase, 2003). MMPs are a large family of zinc-dependent endopeptidases that are capable of cleaving multiple ECM proteins. MMPs can be subdivided into multiple categories depending on their substrate specificity, such as collagenases, stromelysins, and gelatinases (Visse and

Nagase, 2003). MMP2 and MMP9, as well as membrane-type matrix metalloproteinase-1 (MT1-

MMP, also known as MMP14), possess the ability to hydrolyze components of the basement membrane, and have recently emerged as key molecules involved in mediating various aspects of tumor growth and metastasis (Chambers and Matrisian, 1997; Egeblad and Werb, 2002a; Polette et al., 2004;

Shah et al., 2009). Furthermore, MMP2/9 and MT1-MMP are known to stimulate tumor angiogenesis as well as epithelial-to-mesenchymal transition (EMT), in large part through partial ECM proteolysis.

Consistent with these observations, increased expression of MMP2/9 and MT1-MMP has been linked to increased invasiveness in tissue culture cells and increased metastasis in mice (Itoh et al., 1999; Itoh et al., 1998b; Schmalfeldt et al., 2001).

Since increased levels of MMPs correlate with increased metastatic potential of tumors, the mechanisms regulating the production of MMPs have been an active area of research. MMP2/9 and

MT1-MMP protein levels are transcriptionally tightly regulated. In addition to this, the activities of

MMP2 and MMP9 are further controlled by extracellular activation of the pro-enzyme and by inhibition of the active MMPs by extracellular inhibitors, such as tissue inhibitors of MMPs (TIMPs)

(Egeblad and Werb, 2002a). Interestingly, MT1-MMP was shown to cleave and activate pro-MMP2

(Strongin et al., 1995). Finally, MMP2/9 and MT1-MMP undergo regulated targeting and secretion at the sites of forming invadopodia, the actin-rich finger-like cellular projections located at the ventral side of the cell (Murphy and Courtneidge, 2011a; Poincloux et al., 2009b; Stylli et al., 2008). Invadopodia are sites of localized ECM degradation and have been shown to be induced by Src kinase to mediate cancer cell invasion in vitro (Murphy and Courtneidge, 2011a; Murphy and Gavrilovic, 1999). The role of invadopodia during cancer cell invasion in vivo is much less defined, but it has been shown that high expression levels of various invadopodia-forming proteins correlate with an increased metastatic potential (Blouw et al., 2008; Clark et al., 2009; Weaver, 2008a). Furthermore, recent studies have demonstrated the formation of invadopodia-like structures in vivo using intravital imaging (Quintavalle et al., 2010).

Despite the importance of MMP targeting to the invadopodia, the mechanisms regulating subcellular transport of MMPs are only beginning to emerge. MT1-MMP, MMP2 and MMP9 have been shown to be enriched at the invadopodia ( Poincloux et al., 2009; Clark et al., 2008; Nakahara et al.,

1997; Artym et al., 2006; Bourguignon et al., 1998; Monsky et al., 1993). It has been shown that endocytic recycling of MT1-MMP is important in targeting it to the plasma membrane and invadopodia

(Bravo-Cordero et al., 2007; Remacle et al., 2003). Furthermore, it has been demonstrated that selective endocytosis of MT1-MMP also plays a role in regulating its activity towards the ECM (Remacle et al.,

2003). In contrast, almost nothing is known about the membrane transport machinery involved in targeted secretion of MMP2 and MMP9. Intracellular transport and targeting of membrane- bound organelles are regulated by multiple protein families. Rab GTPases have emerged as

19 key regulators of membrane transport and were shown to be required for multiple membrane transport steps, such as cargo sorting, transport and fusion with the donor membranes. Thus, to start identifying the membrane transport and targeting machinery that regulates MMP2/9 secretion, we performed a Rab GTPase siRNA library screen. This screen identified Rab40b as a small monomeric GTPase required for the secretion of both, MMP2 and MMP9. We have shown that, unlike

MT1-MMP, MMP2 and MMP9 secretion is not dependent on endocytic transport, but instead relies on transport from the trans-Golgi Network (TGN) via VAMP4 and Rab40b-containing secretory vesicles.

Rab40b knock-down results in mistargeting of MMP2 and MMP9 to lysosomes, where they are degraded. We also demonstrate that Rab40b mediates MMP2/9 targeting to invadopodia and is required for invadopodia-dependent ECM degradation. Finally, we show that Rab40b knock-down inhibits in vitro invasion of MDA-MB-231 cells, while having no effect on cell motility. Based on these findings, we propose that Rab40b is the key GTPase required for MMP2/9 intracellular transport and targeting to the newly formed invadopodia, thus affecting the invasive capacity of breast cancer cells.

Materials and Methods

Antibodies and Constructs

Mouse monoclonal anti-myc (9E10) antibodies were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Mouse anti-MMP2 and mouse anti-MMP9 used in the

ELISA screen were purchased from Millipore Corp. (Temecula, CA). Anti-myc antibody conjugated with HRP (GTX21261) was obtained from Gene Tex Inc. Anti-FLAG antibody was purchased from Sigma (St. Louis, MO). Rabbit polyclonal anti-Tks5 antibody was generated using recombinant human Tks5-SH3-1/Tks5-SH3-4 domains as previously described (Blouw et al., 2008; Seals et al., 2005). Anti-VAMP4 antibodies were previously

20 described (Gordon et al., 2010). Alexa594 and Alexa488-conjugated anti-rabbit and anti- mouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories

(West Grove, PA).

Cell culture and generation of tet-inducible MDA-MB-231 cell lines

MDA-MB-231 cells were cultured in 50% RPMI-1640 and 50% DMEM with 4.5 g/L glucose, 5.84 g/L L-glutamine and 10% heat inactivated fetal bovine serum (FBS), and supplemented with 100 IU/ml penicillin and 100 g streptomycin. To create tet-inducible

MDA-MB-231 MMP2-myc and MMP9-myc stable cell lines, MMP2-myc or MMP9-myc were cloned into the pHUSH retroviral expression vector (Genentech, South San Francisco,

CA). Stable, clonal cell lines were then selected using 1 g/ml of puromycin and grown using the above described media (MDA-MB-231 media supplemented with tet-free FBS

(Clontech Laboratories, Mountain View, CA)). To induce either MMP2-myc or MMP9-myc expression, stable cells lines were incubated in the presence of 1 g/ml of doxycycline for 24 hrs. siRNA library screen

The siRNA screen was performed with a pool of 4 different siRNAs against each

RabGTPase in the library. MDA-MB-231 stable cell lines expressing either MMP9-myc or

MMP2-myc were plated on 96-well plates at a density of 1.2x104 cells and co-transfected with siRNA oligonucleotides using RNAi Max (Invitrogen, Carlsbad, CA), as described in the manufacturers protocol. Transfected cells were incubated for 36 hrs. Complete growth media was then replaced with serum-free media and cells incubated for another 32 hrs. The cell viability was monitored by Perkin Elmer Envision reader using Cell Titer Glow reagent

(Promega, Madison, WI). Media was then collected and levels of secreted MMP2-myc or

MMP9-myc analyzed by ELISA.

ELISA assays

For the siRNA screen, 96-well microplates were coated with 100 L of diluted anti-

MMP2 (2 g/mL in PBS) or diluted anti-MMP9 (0.5 g/mL in PBS) antibody solution. This was followed by a wash after which plates were blocked with 200 L of blocking buffer.

Plates were then washed and incubated with 100 L of media collected from siRNA-treated cells. After 2 hrs of incubation, plates were washed and incubated with 90 L of mouse anti- myc antibody conjugated to HRP. Plates were again incubated for 1 hour at room temperature (22oC), followed by a wash. The amount of MMP2-myc or MMP9-myc was measured using HRP substrate solution and absorbance at 450 nm using NanoDrop ND-1000

Spectrophotometer.

To measure levels of secreted IL-6, mock or Rab40b siRNA-treated cells were seeded into 6-well dishes at a density of 8x105 cells. IL-6 secretion was induced with 1 mg/ml of

LPS. After incubation for 16 hrs, media was collected for ELISA analysis and cells were harvested for Bradford assay to normalize samples. IL-6 ELISA analysis was performed using IL-6 DuoSet ELISA Development system as described in the manufacturer’s protocol.

The absorbance at 450 nm was determined using NanoDrop ND-1000Spectrophotometer.

In vitro invasion and motility assays

MDA-MB-231 cell motility or invasion was measured using Transwell filter assays.

To measure motility, mock or siRNA-treated cells were resuspended in fresh serum- containing media and added to the top chamber of six Transwell filter insets (6.5 mm filters with 8 m pores, Corning Inc., Corning, NY) at a density of 100,000 cells per filter. MDA-

MB-231 conditioned media (the media after 48 hr incubation with cells) was added to the bottom chamber. In invasion assays, filter was coated with Matrigel matrix. After a 16 hr incubation (invasion assays) or 8 hour incubation (motility assays) cells were stained with

0.1% crystal violet. After three washes, cells that remained on the upper surface of the filter were removed with a cotton swab. The dye from cells remaining on the bottom side of the filter was extracted with 2.5% acetic acid and quantified by measuring optical density at 570 nm.

Fluorescent microscopy

MDA-MB-231 cells were plated on collagen-coated glass cover slips, grown overnight and fixed with 4% paraformaldehyde for 15 min, permeabilized for 10 min in phosphate buffered saline (PBS) containing 0.4% saponin, and non-specific sites were blocked with PBS containing 0.2% bovine serum albumin and 1% fetal bovine serum. Cells were then incubated with specific antibodies, washed in PBS, and mounted in VectaShield

(Vector Laboratories, Burlingame, CA). Cells were imaged with an inverted Zeiss Axiovert

200M deconvolution microscope with a 63x oil-immersion lens and Sensicam QE CCD camera. Image processing was performed using Intelligent Imaging Innovations (Denver,

CO) three-dimensional rendering and exploration software.

To visualize invadopodia, Transwell filter insets (6.5 mm filters with 8 m pores,

Corning Inc., Corning, NY) were coated with Matrigel. Briefly, filters were coated with 60 l

o of Matrigel for 1 hour at 4 C, after which 55 l of Matrigel was removed and filters were incubated at 37oC for 30 min to allow Matrigel to polymerize. Media was then added to both sides of the filter for 15 min to rehydrate the Matrigel. MDA-MB-231 cells stably expressing

FLAG-Rab40b were then seeded at a density of 50,000 cells per filter. MDA-MB-231

23 conditioned media (the media after 48 hr incubation with cells) was added to the bottom chamber. Cells were incubated for either 24 or 36 hours, fixed and stained with rhodamine- phalloidin, anti-myc or anti-FLAG antibodies. Z-stacks of invading cells were imaged using

100 nm z-steps. The 3D rendering was performed using Intelligent Imaging Innovations

(Denver, CO) three-dimensional rendering and exploration software.

FACS analysis

Mock or Rab40b siRNA-treated MDA-MB-231 cells were incubated at 37oC for 64 hrs. Cells were then trypsinized and fixed with 4% paraformaldehyde, followed by quenching with 0.1 M glycine. Cells were then permeabilized with FACS buffer (PBS containing 0.4% saponin, 1% BSA and 2% FBS) for 30 min, followed by incubation with primary antibodies for 1 hr. Cells were washed three times with FACS buffer and incubated for 30 min with

FITC-conjugated secondary antibodies. After another set of three washes, cells were resuspended in 500 mL of PBS and analyzed by flow cytometry using Cytomics FC500 flow cytometer (Beckman Coulter, Brea, CA).

To analyze plasma membrane MMP14, mock, Rab40b siRNA or VAMP4 siRNA- treated MDA-MB-231 cells were incubated at 37oC for 64 hrs. Cells were then trypsinized and resuspended in Hepes-buffered and serum-supplemented media. Cells were then incubated with anti-MMP14-APC antibodies at 37oC for 1 hr. Cells were washed three times with media, resuspended in 500 mL of PBS and analyzed by flow cytometry using Cytomics

FC500 flow cytometer (Beckman Coulter, Brea, CA).

Zymography assays

Mock or Rab40b siRNA-treated MDA-MB-231 cells were incubated in the complete media at 37oC. After 48 hrs of incubation, media was replaced with Opti-MEM (Invitrogen,

Carlsbad, CA) and cells were incubated at 37oC for another 24 hrs. Opti-MEM media was collected and cell lysates were harvested using PBS containing 1% Triton X100. The levels of secreted MMP2 and MMP9 in Opti-MEM were then analyzed by zymography. Fetal

Bovine serum (rich in MMP2 and MMP9) was used as positive control. Opti-MEM collected from cell-free well was used as negative control. Briefly, Opti-MEM samples were diluted in the standard SDS-PAGE sample buffer without a reducing agent. Samples were then separated using 7.5% polyacrilamide gels containing 3 g/mL of gelatin. Sample loading was normalized based on cell lysate protein concentrations. Following electrophoresis, gels were washed twice with 2.5% Triton X100 to remove SDS. Gels were then incubated in digestion

o buffer (50 mM Tris, pH 8.0 containing 5 mM CaCl2) at 37 C for 24-48 hrs. Gels were subsequently stained with Coomassie Brilliant Blue dye. The extent of gelatin proteolysis by endogenous MMP2 (bottom band) and MMP9 (top band) was detected as white bands on a dark background.

In situ zymography assays

The in situ zymography/matrix degradation assay was done by coating 18mm round coverslips in 12 well plates with 2.5% gelatin/2.5% sucrose in PBS at 37oC. The gelatin was allowed to set at 4oC before cross-linking with 0.5% glutaraldehyde by incubation at 4oC for

15 minutes. A 50 µg/mL solution of FITC-fibronectin (Cytoskeleton Inc., Denver, CO) was then overlaid on top of cross-linked gelatin and incubated in the dark for 1 hour at 4oC. The dish was sterilized with 70% ethanol, washed with DMEM, and equilibrated with

25 invadopodia medium (DMEM supplemented with 20% FBS (Atlanta Biologicals) and 10%

Nu-Serum) for 30 min. MDA-MB-231 cells (4x104cells in 2 mL of invadopodia medium) were then added to each well and incubated for 20 hrs. The cells were then fixed in 3% paraformaldehyde, permeabilized with 0.4% Triton X-100 in PBS and stained with rhodamine-phalloidin (Invitrogen, Carlsbad, CA). To quantify invadopodia formation and localized matrix degradation, ten randomly chosen fields were imaged (using 63x objective) per treatment for each experiment. A total of 260-330 cells were counted in at least three independent experiments. To measure the number of cells with invadopodia, cells were counted based on the presence of actin puncta and degradation spots seen underneath the cells within the cell boundaries. To measure invadopodia-associated area of degradation, the areas lacking FITC-fibronectin fluorescence were measured using Intelligent Imaging

Innovations (Denver, CO) three-dimensional rendering and exploration software. Only degradation areas associated within cell boundaries were analyzed.

Reverse transcriptase polymerase chain reaction (RT-PCR) and quantitative PCR (qPCR)

Total RNA was extracted from 2x107 MDA-MB-231 cells using TRIzol (Invitrogen,

Carlsbad, CA) according to manufacturer’s protocol. Reverse transcription to cDNA was performed with SuperScript III (Invitrogen, Carlsbad, CA) using random hexamer primers.

PCR was performed using Taq polymerase (Invitrogen, Carlsbad, CA). To quantify the percent of knock-down, cDNA from mock or siRNA-treated cells was analyzed in triplicate by qPCR amplification using Sybr Green qPCR Master Mix using Applied Biosystems

ViiA7 Real-Time PCR System. The qPCR amplification conditions were: 50oC (2 min), 95oC

(10 min), 40 cycles at 95oC (15 sec), 60oC (1 min). Primer pairs were designed to amplify mRNA-specific fragments and unique products were tested by melt-curve analysis. Relative

26 quantification was calculated by the CT method. Data shown as the fold change (averaged from two independent experiments) in Rab40b knock-down cells as compare to mock cells.-actin served as normalizing control (sense- 5’-AAAGACCTGTACGCCAACAC-3’; anti-sense- 5’-GTCATACTCCTGCTTGCTGA-3’). MMP2 was amplified using following primers: sense- 5’-CCTGATGGCACCCATTTACACC-3’; anti-sense- 5’-

CGACGGCATCCAGGTTATCG-3’. MMP9 was amplified using following primers: sense-

5’-CCCTTCTACGGCCACTACTGTG-3’; anti-sense- 5’-GCACTGCAGGATGTCATAG-

3’.

Flow cytometry-based GFP-hGH secretion assay

To measure hGH secretion we used HeLa cells stably expressing GFP-FKBP-hGH as previously described (Gordon et al., 2010) (Fig. 2.2H). This assay is based on the property of

FKBP to form ligand-reversible aggregates, thus trapping GFP-FKBP-hGH in the ER.

Addition of FKBP ligand AP21998 results in solubilization of these aggregates and synchronous secretion of GFP-hGH from the cells. Since hGH is tagged with GFP (Gordon et al., 2010), secretion can be quantified in live cells by measuring the cell –associated GFP fluorescence (not secreted) by flow cytometry.

To measure the effect of Rab40b knock-down on hGH secretion, HeLa cells stably expressing GFP-FKBP-hGH were plated at 25% confluence on a 6 well plates and transfected with siRNA targeted to either Rab40b or Syntaxin 5. Syntaxin 5 siRNA was used as a positive control, since it was previously reported that Syntaxin 5 depletion inhibits hGH secretion (Gordon et al., 2010). 72 hours after transfection, cells were trypsinised and counted before plating at 50,000 cells per well on a 24 well plate. Cells were incubated overnight before use in the secretion assay. Secretion of the GFP-FKBP-hGH was initiated

27 using AP21998 at a concentration of 1 μM in prewarmed culture media. To halt secretion, samples were placed on ice, washed with cold PBS and trypsinized on ice for 2 hrs. To measure the amount of cargo remaining in cells after secretion, samples were analysed using a BD Fortessa flow cytometer with live cells gated by forward and side scatter. Ten thousand live cells were analyzed for each sample. The mean GFP ﬂuorescence was calculated using the software FLOWJO (TreeStar). The percentage of intracellular GFP remaining after secretion was calculated using 3 control unsecreted wells and 3 experimental secreted wells for each condition.

Statistical analyses

Two-tailed independent Student’s t-test was used to analyze the results of MMP2/9 and IL6 secretion, FACS analysis, invasion and motility assays. All statistical calculations were done using GraphPad Prism 5.0d software (La Jolla, CA). Unless otherwise noted, all data shown are the means and standard deviations of three independent experiments.

Results

Rab40b GTPase is required for MMP2 and MMP9 secretion

Since little is known about the regulation of intracellular MMP2 and MMP9 transport, in this study we screened for Rab GTPases that regulate MMP2/9 transport and secretion. To that end, we created tet-inducible MDA-MB-231 cell lines expressing either MMP2-myc (MDA-

MMP2-myc) or MMP9-myc (MDA-MMP9-myc). As shown in Fig. 2.1 A-B, MDA-MMP2- myc and MDA-MMP9-myc cells express and secrete enzymatically active MMP2-myc and

MMP9-myc in a doxycycline dependent manner. Furthermore, the addition of doxycyline increased ECM degradation (Fig. 2.1C) and invasion in these cells (Fig. 2.1D). We next

28 analyzed subcellular localization of MMP2-myc and MMP9-myc. As expected of secretory proteins, MMP2/9-myc were enriched at the perinuclear region (Fig. 2.1E-F a-b), where they colocalized with the trans-Golgi network (TGN) marker VAMP4 (Fig. 2.2). MMP2/9-myc- containing organelles were also found in the cytosol, especially in close proximity to the basal plasma membrane (Fig. 2.1E-F c-d). Taken together, the above data suggest that these cells likely transport and secrete myc-tagged MMP2/9 in a manner similar to endogenous

MMP2/9.

Next, we used an ELISA-based siRNA screen to identify Rab GTPases that regulate secretion of MMP2-myc and MMP9-myc (Fig. 2.3). In all cases, a pool of four different siRNAs was used (for more detailed description of the screen, see Materials and Methods).

Rab GTPases that either increased or decreased MMP2-myc or MMP9-myc secretion were then re-screened using four individual siRNAs. Only candidates that affected MMP2/9 secretion after treatment with at least two out of four siRNAs, were considered for further characterization. As shown in Fig. 2.3, the screen identified several Rab GTPases that either increased or decreased MMP2-myc or MMP9-myc secretion. Interestingly, while most of the identified candidate proteins affected either MMP2-myc or MMP9-myc secretion, only

Rab25 and Rab40b knock-down decreased secretion of both the MMPs. Rab25 was previously identified as a GTPase that regulates cancer cell motility and invasion (Cheng et al., 2006; Cheng et al., 2004). In contrast, little is known about the function of Rab40b.

Therefore, we focused on understanding the cellular function of Rab40b GTPase for the remaining part of this study.

Figure 2.1 Characterization of MDAMB-231 cell lines expressing tetinducible MMP2–Myc or MMP9– Myc. (A,B) MDA-MB- 231 cells expressing doxinducible MMP2–Myc or MMP9–Myc were incubated in Opti-MEM for 24 hours in the absence or presence of 1 g/ml doxycycline. Opti- MEM medium was then collected and the amount of secreted MMP2–Myc or MMP9–Myc was analyzed by (A) immunoblotting or (B) zymography. (C) MDA- MB-231 cells expressing doxinducible either MMP2–Myc or MMP9– Myc were plated on gelatin and fibronectin- HiLyte Fluor488-coated coverslips and incubated in the presence or absence of 1 g/ml doxycycline. After incubation for 20 hours, cells were fixed and invadopodia associated ECM degradation was analyzed by in situ zymography. Data shown are the means and s.e. of three independent experiments. (D) MDA-MB-231 cells expressing dox-inducible MMP2–Myc or MMP9–Myc were plated on matrigelcoated 8-m-pore filters and incubated in the presence or absence of 1 g/ml doxycycline. The ability of cells to invade through Matrigel matrix was analyzed using Crystal Violet staining. The data shown are the means and s.d.from three independent experiments. (E,F) MDA-MB-231 cells expressing (E) MMP2–Myc or (F) MMP9–Myc were fixed and stained with Rhodamine-phalloidin and mouse anti-Myc antibodies. Panels a and b show optical sections at the TGN level, whereas panels c and d show optical sections at the coverslip level. Arrows indicate cytosolic organelles containing MMP2–Myc or MMP9– Myc. Scale bar: 5 m.

Figure 2.2. VAMP4 is present on MMP2/9 transport organelles. (A-F) MMP2-myc (A-C) or MMP9-myc (D-F) expressing MDAMB-231 cells were fixed and co-stained using anti- VAMP4 (B, C, E, F) or anti-myc (A, C, D, F) antibodies. Scale bars 10 m. (G) MDA-MB- 231 cells were treated with DMSO (control), GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/9 inhibitor). The media was then collected and MMP2/9 activity analyzed by zymography. (H) To test whether Rab40b is required for hGH secretion HeLa cells stably expressing GFP-hGH were transfected with either Rab40b or syntaxin 5 siRNAs. After 72 hour incubation cells were treated with AP21998 (1 m) for 80 min at 37oC to allow the exit of GFP-hGH from ER and secretion. The mean flurescence before and after AP21998 treatment was determined by flow cytometry. The data shown are expressed as a percentage of fluorescence before AP21998 treatment (100%) and are means and standard deviations derived from three experiments. The schematics above the bar graph show the GFP-hGH reporter construct used in these experiments.

Figure 2.3. MMP2 and MMP9 secretion regulating proteins identified from siRNA screen. ELISA-based quantification of the secreted MMP2–Myc (A) and MMP9–Myc (B) in cells treated with various Rab siRNAs. For more details see the Materials and Methods. Data shown are the means and s.d. of three independent experiments. All the values above top grey line or below bottom grey line are significantly different from the control at P<0.01 (marked with *). The black bars indicate the Rabs that decreased secretion of both MMP2- Myc and MMP9-Myc.

Rab40b is required for MMP2/MMP9 sorting and secretion

All Rab GTPases function by binding and recruiting their respective effector proteins to the membrane-bound organelles (Pfeffer, 2001). There is very limited information about the localization and function of Rab40b. Rab40b belongs to a sub-family of Rab40 GTPases, that includes two other closely related members, Rab40a and Rab40c, and is characterized by the presence of a SOCS box at their C-terminal half (Fig. 2.4A) (Piessevaux et al., 2008; Stein et al., 2012). Interestingly, depletion of Rab40a did not have any effect on MMP2-myc or

MMP9-myc secretion. In contrast, Rab40c knock-down decreased secretion of MMP2-myc, suggesting that there may be some functional overlap between Rab40b and Rab40c GTPases.

To validate the role of Rab40b in regulating MMP2/9 secretion, we tested the effects of individual siRNAs on MMP2-myc and MMP9-myc secretion. As shown in Fig. 2.4B, all four

Rab40b siRNAs resulted in decrease of Rab40b mRNA levels. Consistent with Rab40b involvement in regulating MMP2/9 intracellular transport, all four siRNAs also decreased the secretion of MMP2-myc and MMP9-myc (Fig. 2.4C and D). Similarly, Rab40b depletion also decreased secretion of endogenous MMP2 and MMP9 (Fig. 2.4E), but not MT1-MMP

(Fig. 2.4F). Depletion of Rab40b also had little effect on LPS-induced release of IL6 (Fig.

2.4G) or GFP-hGH secretion (Fig. 2.2H) suggesting that one Rab40b function is to regulate

MMP2/9 intracellular transport.

Since Rab40b depletion leads to a decrease in MMP2 and MMP9 secretion, one would predict that this would result in the accumulation of MMP2 and MMP9 within the cell.

To determine this, we measured the intracellular levels of MMP2-myc and MMP9-myc using

FACS analysis. Surprisingly, Rab40b knock-down decreased intracellular MMP2/9 levels

(Fig. 2.5A and B), while having no effect on levels of other post-TGN proteins, such as

Figure 2.4. Rab40b knockdown decreases MMP2 and MMP9 secretion in MDAMB- 231 cells. (A) Schematic representation of Rab40b domain structure. (B) The efficiency of Rab40b knockdown as determined by qPCR. (C,D) MDA- MB-231 cells expressing MMP2–Myc (C) or MMP9–Myc (D) were transfected with four different Rab40b siRNAs. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of medium for 24 hours. Medium was then collected and the effect of Rab40b knockdown on secretion of MMP2–Myc and MMP9–Myc analyzed by western blotting. Data shown are the means and s.d. of three independent experiments. (E) MDA-MB-231 cells were transfected with Rab40b siRNA#2. Two days later, equal number of cells were plated in six-well dishes and incubated with 1 ml of Opti-MEM for 24 hours. Opti-MEM was then collected and the effect of Rab40b knockdown on secretion of endogenous MMP2 and MMP9 was analyzed by zymography. Fetal bovine serum (rich in secreted MMP2/9) in the first lane was used as a positive control. Opti-MEM collected from a six-well dish without cells was used as negative control. (F) Mock-,Rab40b-siRNA- or VAMP4-siRNAtreated MDA-MB-231 cells were harvested and analyzed by flow cytometry to measure the levels of endogenous plasma membrane MT1-MMP. Data shown are the means and s.d.of three independent experiments. (G) Mock- or Rab40b-siRNA-treated MDA-MB-231 cells were plated in six well dishes and stimulated with 1 mg/ml of LPS. After incubation for 16 hours,medium was collected and the levels of secreted IL-6 analyzed using ELISA. The data shown are the means and s.d. of three independent experiments.

Figure 2.5. Rab40b increases lysosomal degradation of MMP2 and MMP9. (A–C) Mock- or Rab40b-siRNA-treated MDAMB-231 cells were harvested and analyzed by flow cytometry to measure the levels of intracellular MMP2–Myc (A), MMP9– Myc (B),transferrin receptor (C) and CD63 (C). Data shown in A and B are the means and s.d. of three independent experiments. Data shown in C are the means of two independent experiments. (D) MDA-MB-231 cells stably expressing MMP2– Myc or MMP9–Myc were transfected with Rab40b siRNA. Two days later, cells were incubated in the presence of absence of bafilomycin for 12 hours and medium was collected to measure the amounts of secreted MMP2–Myc and MMP9–Myc (inset). Cells were then analyzed by flow cytometry to measure the levels of intracellular MMP2–Myc and MMP9– Myc. Data shown are the means and s.d. of three independent experiments. (E,F) MDA-MB- 231cells stably expressing MMP2–Myc (E) or MMP9–Myc (F) were transfected with Rab40b siRNA. Two days later, cells were treated with bafilomycin for 12 hours, then fixed and costained with anti-Myc (red) or anti-CD63 (green) antibodies. Nuclei are stained blue with DAPI.Scale bar: 5 m.

35 transferrin receptor, CD63 or IL6 (Fig. 2.5C and 2.4G). Since Rab40b depletion resulted in simultaneous decrease in MMP2/9 secretion and intracellular levels, it raises the possibility that in Rab40b-depleted cells MMP2/9 may be mis-sorted to lysosomes and degraded. To test this hypothesis, we incubated Rab40b siRNA-treated cells in the presence or absence of the lysosomal inhibitor bafilomycin. As shown in figure 2.5D, bafilomycin treatment reversed the effects of Rab40b siRNA on intracellular levels of MMP2 and MMP9. Interestingly, bafilomycin did not rescue the Rab40b-depletion induced secretory block, suggesting that in addition to MMP2/9 sorting, Rab40b may also be directly affecting the targeting and fusion of MMP2/9 secretory vesicles with the cellular plasma membrane. To further test this possibility, we stained Rab40b siRNA transfected and bafilomycin-treated cells with anti- myc and anti-CD63 (lysosomal marker) antibodies. As shown in Fig. 2.5E-F, MMP2-myc and MMP9-myc were now present in the small organelles scattered through the cytosol as well as in bafilomycin-induced enlarged lytic organelles (see arrows).

Rab40b is localized to VAMP4-containing secretory vesicles

Subcellular protein localization can often provide clues about their cellular function.

To this end, we compared the localization of FLAG-tagged Rab40b to the localization of

VAMP4, a well-established marker for secretory vesicles and TGN (Steegmaier et al., 1999).

As shown in Fig. 2.6A-F, FLAG-Rab40b was present on VAMP4-containing vesicles located at the edges of the TGN (marked by arrows). Although, the resolution of the images does not allow us to unequivocally determine the identity of these organelles, it is possible that these are VAMP4-containing secretary vesicles budding from the TGN. Consistently, FLAG-

Rab40b colocalizes with VAMP4-secretory vesicles at the periphery of the cell (Fig. 2.6G-L, arrows), (Kakhlon et al., 2006; Krzewski et al., 2011; Steegmaier et al., 1999). Similarly,

Figure 2.6. FLAG–Rab40b colocalizes withVAMP4-containing secretory vesicles. MDA- MB-231 cells were transfected with FLAG–Rab40b and plated on collagen-coated glass coverlips. Cells were then fixed and stained with anti-FLAG (A,C,D,F,G,I,J,L,M,O), anti- VAMP4 (B,C,E,F,H,I,K,L) and anti-FIP1(N,O) antibodies. In D–F, arrows indicate VAMP4 and Rab40b-containing vesicles at the edges of the TGN. In J–L, arrows indicate peripheral organelles containing VAMP4 and Rab40b. In C and I, boxed region marks area shown as higher magnification images in D–F and J–L. Nuclei in C,F,I are stained blue with DAPI.

VAMP4 also colocalized with MMP2-myc and MMP9-myc (Fig. 2.2A-F). Since MT1-MMP was shown to be present at recycling suggesting that Rab40b organelles contain VAMP4 endosomes (Bravo-Cordero et al., 2007), we next compared the localization of FLAG-

Rab40b and Rab11-FIP1/RCP, a known recycling endosome marker (Peden et al., 2004). As shown in Fig. 2.6M-O, FLAG-Rab40b was not present on recycling endosomes, suggesting that MMP2/9 and MT1-MMP are probably targeted to the plasma membrane via distinct membrane transport pathways. Consistent with this, depletion of various Rab11 effector proteins (known to regulate recycling endosomes) or the Exocyst complex (known to mediate

MT1-MMP targeting) did not have any effect on MMP2/9 secretion (data not shown). Our data suggests that VAMP4 may be the R-SNARE responsible for fusion of MMP2/9 secretory vesicles with the plasma membrane. To examine the role of VAMP4, we analyzed the effect of VAMP4 knock-down (Fig. 2.7A) on secretion of endogenous MMP2 and

MMP9. As shown in Fig. 2.7B-D, VAMP4 depletion reduced the amounts of MMP2 and

MMP9 in media. In contrast, the depletion of other R-SNAREs, namely VAMP3 and

VAMP7, did not have any effect on MMP2/9 secretion (Fig. 2.7A-D).Previous findings from several laboratories have demonstrated that MMP2 and MMP9 are transported to the forming invadopodia during cancer cell invasion (Murphy and Courtneidge, 2011a; Poincloux et al.,

2009b). Thus, we examined whether Rab40b-containing vesicles are also present at invadopodia. To visualize invadopodia, FLAG-Rab40b-expressing MDA-MB-231 cells were seeded on Matrigel-coated Transwell filters containing 8 m pores (for more details see

Methods). After 24 or 36 hours, cells were fixed and stained with rhodamine-phalloidin to visualize cells at different stages of invasion through Matrigel and filter pores (Fig. 2.8A).

During early stages of invasion cells formed an actin rich invadopodia at the ventral side of

Figure 2.7. VAMP4 is required for MMP2/9 secretion. (A) Control, VAMP4, VAMP3 or VAMP7 siRNA treated MDA-MB-231 cells were incubated for 72 hrs. Cellular lysates were then immunoblotted for the presence of VAMP4, VAMP3, VAMP7 and transferring receptor. (B-D) Control or VAMP siRNA treated MDA-MB-231 cells were incubated with serum-free Opti-MEM for 4 hrs. Media was then collected and the activity of secreted MMP2 and MMP9 was analyzed by zymography. In panel (C), increasing volumes of collected media was loaded. Panel (D) shows quantification of secreted MMP9. The data shown are the means and standard deviations calculated from three independent experiments. (E) Control or VAMP4 siRNA-treated cells were plated on Matrigel matrix coated Boyden chambers. The invasion of cells from the top to the bottom chamber was then analyzed. The data shown are the means and standard deviations calculated from three independent experiments.

Figure 2.8. Localization of Rab40b containing organelles during cell invasion in vitro. MDA-MB-231 cells stably expressing FLAG–Rab40b were seeded on Matrigel-coated filters containing 8 m pores. Cells were incubated for either 24 hours (B,D) or 36 hours (F). Cells were then fixed and stained with Rhodamine-phalloidin or anti-FLAG antibodies. Drawings in A depict the invasion stages imaged in panels B,D,F. Arrows in all images indicate invadopodia or pseudopodia (probably derived from invadopodia). B,D,F are 3D rendering of images shown in C,E,G and show cells from Z-Y (left panels) and X-Y (right panels) planes.Lines in D,F indicate the level of the optical sections depicted in E,G.

40 the cell (Fig. 2.8A-C). FLAG-Rab40b-containing organelles were also observed to be enriched at the site of forming invadopodia (Fig. 2.8C, arrow). At the mid-stage of the invasion, invadopodia extended into the filter pore (Fig. 2.8 D) and also contained Rab40b- organelles clustered close to the invadopodia plasma membrane (Fig. 2.8E). Finally, these invadopodia elongated and filled the entire filter pore, as cells started migrating through the filter to the bottom chamber (Fig. 2.8F-G). Similarly, MMP2-myc and MMP9-myc organelles also accumulated within the invadopodia during cell invasion through the 8 m pore (Fig. 2.9). Taken together, all these data suggest that Rab40b-containing vesicles could mediate the delivery of the MMP2/9 to the invadopodia during cancer cell invasion.

Rab40b is required for breast cancer cell invasion and invadopodia-dependent ECM degradation in vitro

Previous research has suggested that MMP2 and MMP9 play a key role in degradation of the ECM during breast cancer cell metastasis. Thus, we tested the effect of

Rab40b knock-down on the ability of MDA-MB-231 cells to invade in vitro using Transwell filters (with 8 m pores) coated with Matrigel. Consistent with the involvement of Rab40b in

MMP2/9 secretion, Rab40b, but not Rab40a, depletion resulted in significant decrease in cell invasion, while having no effect on cell motility (Fig. 2.10A-B). Since VAMP4 is present on

Rab40b-containing organelles, we also tested the effect of VAMP4 depletion on cell invasion. As shown in Fig. 2.7E, VAMP4 depletion inhibited the invasion of MDA-MB-231 cells. Our cellular localization data suggested that Rab40b is involved in targeting MMP2/9 vesicles to the invadopodia. To test this hypothesis, we investigated whether Rab40b is required for invadopodia-associated MMP secretion and ECM degradation. MDA-MB-231 cells were

Figure 2.9. MMP2-myc and MMP9-myc accumulate within the invadopodia during cell invasion. (A) Schematic representation of cells in mid-invasion stage that are imaged in (B- H). (B-H) MDA-MB-231 cells expressing MMP2-myc (B-E) or MMP9-myc (F-H) were plated on Matrigel-coated filters containing 8 m pores. Cells were incubated for 24 hours, then fixed and stained with anti-myc antibodies. Z-stack images with the 200 nm step size were then taken. Panels (B and C) are 3D rendering of images and show cells from Z-X (C) and X-Y (B) planes. Line in panel (B) marks the level of the optical sections depicted in panels (C-E). Panels (C-E) show MMP2-myc organelles present in the invasion pore. Panels (F-H) show MMP9-myc organelles present in the invasion pore.

Figure 2.10. Rab40b is required for MDA-MB-231 cell invasion in vitro. (A,B) Mock- or siRNA-treated MDA-MB-231 cells were plated on matrigel-coated (A) or uncoated (B) 8- mm-pore filters. Cells were then incubated for 8 hours (B) or 16 hours (A) and the extent of cell migration to the bottom side of the filter was analyzed by Crystal Violet staining (see the Materials and Methods). Data shown are the means and s.d. of three independent experiments; *P.0.05. (C–H) MDA-MB-231 cells were plated on gelatin and fibronectin-HiLyte Fluor488-coated coverslips (D,E,G and H). After incubation for 20 hours, cells were fixed and stained with Rhodamine-phalloidin (C,E,F,H). Arrows indicate invadopodia. Scale bars: 5 m (C–E), 1 m (F–H). (I–K) MDA-MB-231 cells were plated on gelatin-coated coverslips. After incubation for 20 hours, cells were fixed and stained with Rhodamine-phalloidin (I,K) and rabbit anti-Tks5 antibodies (J,K). Scale bar: 1 m.

43 plated on gelatin-coated glass coverslips overlaid with fibronectin conjugated to HiLyte

Fluor488. As shown in Fig. 2.10C-H, MDA-MB-231 cells formed actin-rich punctate structures, which associated with localized degradation of the fibronectin/gelatin matrix.

These actin puncta can be identified as invadopodia, since they contain the known invadopodia marker Tks5 (Fig. 2.10I-K) (Courtneidge et al., 2005a; Murphy et al., 2011).

Next, we tested whether Rab40b is required for invadopodia formation and invadopodia- associated ECM degradation. Rab40b depletion resulted in a decrease in invadopodia- associated ECM degradation (Fig. 2.11A-G), but had no effect on the cell’s ability to form invadopodia (Fig. 2.12).

While Rab40b knock-down decreased invadopodia-associated ECM degradation, it remains unclear whether this effect is due to a decrease in MMP2/9 targeting. Some reports have questioned whether MMP2/9 are required for invadopodia-associated ECM degradation, or whether invadopodia are dependent only on MT1-MMP activity (Hotary et al., 2006; Poincloux et al., 2009b). In order to validate the role of MMP2/9 in invadopodia- associated ECM degradation, cells were treated with a broad-spectrum MMP inhibitor

GM6001 or MMP2/9 specific inhibitor SB3CT (Fig. 2.11H and Fig 2.12 A-D). As previously reported (Clark et al., 2007), the broad-spectrum MMP inhibitor GM6001 almost completely blocked invadopodia-associated ECM degradation, while having no effect on invadopodia number (Fig. 2.11H; Figure 2.12B and F). In contrast, treatment with MMP2/9 –specific inhibitor SB3CT (Ikejiri et al., 2005; Overall and Kleifeld, 2006) only partially inhibited invadopodia induced ECM degradation (Fig. 2.11H; Figure 2.12C and F). Taken together, these data indicate that in MDA-MB-231 cells, MMP2/9 secretion is at least partially responsible for invadopodia-dependent ECM degradation.

Figure 2.11. Rab40b is required for invadopodia- associated ECM degradation.(A–F) Untreated or Rab40b- siRNA-treated MDA-MB- 231 cells were plated on gelatin and fibronectin HiLyte Fluor488-coated coverslips (D–F). After 20 hours of incubation, cells were fixed and stained with Rhodamine-phalloidin (A– C). Scale bars: 25 m. (G) Quantification of ECM degradation in untreated and Rab40b siRNA treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (H) Quantification of ECM degradation in MDA-MB- 231 cells treated with either GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/MMP9 inhibitor). Where indicated, cells were also treated with Rab40b siRNA. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed. (I) Mock-, Rab40b-siRNA- or VAMP4 siRNA-treated cells were harvested and the levels of mRNA encoding Rab40b analyzed by qPCR (graph). The data shown are the means and s.d. The levels of VAMP4 were analyzed by western blotting (top panels) with tubulin used as a loading control. (J) Quantification of ECM degradation in mock-, Rab40b-siRNA- or VAMP4-siRNA-treated MDA-MB-231 cells. Data shown are the means and s.e. of three independent experiments. n is the total number of cells analyzed.

Figure 2.12. MMP2/9 are required for invadopodia-associated ECM degradation. (A-D) Images of MDA-MB-231 cells treated with either GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/MMP9 inhibitor). Where indicated, cells were also treated with Rab40b siRNA. Cells were plated on gelatin/fibronectin-HiLyte Fluor488 coated coverslips bottom panels. After 20 hr incubation, cells were fixed and stained with rhodamine-phalloidin (top panels). (E) Quantification of invadopodia formation (as determined by counting actin spots) in untreated and Rab40b siRNA-treated MDA-MB-231 cells. Data shown are the means and standard error of three independent experiments. N is the total number of cells analyzed. (F) Quantification of invadopodia formation (as determined by counting actin spots) in MDA- MB-231 cells treated with either GM6001 (broad-spectrum MMP inhibitor) or SB3CT (MMP2/MMP9 inhibitor). Where indicated, cells were also treated with Rab40b siRNA. Data shown are the means and standard error of three independent experiments. N is the total number of cells analyzed.

To further test whether Rab40b is required for MMP2/9 targeting to the invadopodia, we co-treated MDA-MB-231 cells with Rab40b siRNA and SB3CT. As shown in Fig. 2.11H, simultaneous inhibition of MMP2/9 and depletion of Rab40b did not further inhibit ECM degradation as compared to Rab40b knock-down or MMP2/9 inhibition alone (Fig. 2.11H;

Fig. 2.12D and F), demonstrating that Rab40b and MMP2/9 likely act in the same pathway.

Similarly, co-knock-down of Rab40b and VAMP4 also did not further inhibit ECM degradation compared to depletion of Rab40b or VAMP4 alone (Fig. 2.11I-J). These data combined with our imaging experiments suggest that Rab40b and VAMP4 are likely responsible for targeting of MMP2/9-containing transport vesicles to the invadopodia during invasion of cancer cells.

To test whether Rab40b plays a similar role in other cancers, we next tested the effect of Rab40b knock-down on invadopodia-associated ECM degradation in human melanoma

HMCB cells. HMCB cells also formed invadopodia reminiscent to MDA-MB-231 breast cancer cells (Fig. 2.13A-B). Importantly, Rab40b knock-down also led to a decrease in the area of invadopodia associated ECM degradation (Fig. 2.13C-F), suggesting that Rab40b also likely regulates targeted MMP2/9 secretion in non-breast cancers.

Discussion

Multiple studies have demonstrated that MMP2 and MMP9 play an important role during invasion and metastasis of various cancers (Chambers and Matrisian, 1997; Egeblad and Werb, 2002a; Polette et al., 2004; Shah et al., 2009), yet the machinery that regulates

MMP2 and MMP9 transport and fusion with plasma membrane remains elusive. In this study, we used a siRNA screen to identify Rab40b as a small monomeric GTPase that is

Figure 2.13. Rab40b is required for invadopodia-associated ECM degradation in HMCB cells. (A-D) Untreated or Rab40b siRNA-treated HMCB cells were plated on gelatin/ fibronectin-HiLyte Fluor488 coated coverslips (B and D). After 4 hrs of incubation, cells were fixed and stained with rhodamine-phalloidin (A and C). Size bars 10 m. (E) Quantification of ECM degradation in untreated and Rab40b siRNA-treated HMCB cells. Data shown are the means and standard deviations of three independent experiments. N is the total number of cells analyzed. (F) Mock or Rab40b siRNA-treated cells were harvested and the levels of Rab40b mRNA analyzed by qPCR. The data shown are the means and standard deviations.

48 required for secretion of both MMP2 and MMP9. All Rab GTPases act as master regulators of various membrane transport steps, by regulating cargo sorting, vesicle budding, vesicle transport and vesicle targeting to the appropriate acceptor compartment (Pfeffer, 2001).

Prior to our study, little was known about the cellular function of Rab40b GTPase, or the identity of Rab40b interacting proteins. Thus, to further understand the mechanisms of

MMP2/9 transport, we investigated the localization and function of Rab40b during MDA-

MB-231 breast cancer cell invasion in vitro. Interestingly, we have shown that Rab40b- containing vesicles co-localize with R-SNARE, VAMP4, which has been shown to mediate protein transport from the TGN to the plasma membrane (Steegmaier et al., 1999).

Furthermore, we demonstrated that Rab40b and VAMP4 depletion by siRNA results in a decrease in MMP2/9 secretion and MDA-MB-231 cell invasion. The data described above indicate that MMP2/9 are transported to the plasma membrane via secretory vesicles containing Rab40b and VAMP4. Interestingly, MT1-MMP, the other MMP that mediates

ECM degradation during cell invasion, was shown to be transported to the plasma membrane via recycling endosomes (Hotary et al., 2006; Poincloux et al., 2009b). Furthermore, recent work demonstrated that in addition to Rab8, MT1-MMP targeting to plasma membrane requires the Exocyst complex, a protein complex known to function as a transport transport and secretion (Fig. 2). Thus, MMP2/9 and MT1-MMP appear to be transported and secreted via distinct membrane transport pathways.

One of the effects of Rab40b knock-down was a decrease in MMP2/9 secretion. This decrease, at least in part, was caused by increased lysosomal degradation of MMP2 and

MMP9. Since Rab40b localizes to the VAMP4-containing budding sites at TGN, it is a likely

vesicle tethering factor (Sakurai-Yageta et al., 2008). We demonstrate that siRNA-dependent knock-down of Rab8 or of the Exocyst complex components has no effect on MMP2/9 that Rab40b is required for appropriate sorting of MMP2 and MMP9 to the vesicles destined to be transported to the plasma membrane. Interestingly, inhibition of lysosomal degradation did not rescue the MMP2/9 secretion defect, suggesting that Rab40b is also required for

MMP2/9 targeting and fusion with the plasma membrane. Consistent with this observation,

Rab40b knock-down inhibited invadopodia-associated ECM degradation and in vitro invasion of MDA-MB-231 cells, suggesting that Rab40b regulates MMP2/9 sorting at TGN as well as MMP2/9 vesicle targeting to the invadopodia.

Invadopodia are actin-rich, finger-like cellular projections that have been implicated in mediating ECM degradation and cell invasion (Murphy and Courtneidge, 2011a).

Invadopodia have been primarily studied in tissue culture cells, and are sometimes referred to as podosomes. The functional differences between podosomes and invadopodia remain unclear, but generally actin structures in cancer cells are called invadopodia, while in non- cancerous cells they are called podosomes. It has been demonstrated that MMP2/9 and MT1-

MMP are all enriched at the invadopodia where they mediate ECM degradation in vitro and in vivo (Murphy and Courtneidge, 2011a). Interestingly, while Rab40b knock-down only had a moderate effect on MMP2/9 secretion into media, it significantly decreased MMP2/9- dependent invadopodia associated ECM degradation. Taken together, our data suggests that

Rab40b is required for MMP2/9 secretion at the newly formed invadopodia. However, knock-down of Rab40b only decreased ECM degradation at invadopodia by 50%. The rest of the degradation is presumably mediated by MT1-MMP. This is consistent with previous observations that MT1-MMP is transported to invadopodia via Exocyst-dependent recycling

50 endosomes (Sakurai-Yageta et al., 2008). The question that remains is why do cancer cells need two distinct pathways of transporting MMPs to invadopodia? Since VAMP4 and the

Exocyst complex are also present at the plasma membrane outside invadopodia, it is possible that overlapping targeting mechanisms via two different transport pathways is required to ensure the fidelity of ECM degradation at the invadopodia. In addition to degrading collagen,

MT1-MMP has been shown to cleave and activate MMP2 (Strongin et al., 1995). Thus co- targeting of MMP2/9 and MT1-MMP to the same location would result in an amplification of

ECM degradation associated with invadopodia.

While this study demonstrates that Rab40b is required for MMP2/9 sorting and targeting to invadopodia, it is still unknown what effector proteins bind to Rab40b. All Rab

GTPases function by recruiting multiple effector proteins, which then mediate transport vesicle formation, transport and targeting (Pfeffer, 2001). Furthermore, Rab40b has a SOCS box domain, a feature unique to the Rab40 sub-family of proteins (Stein et al., 2012).

Interestingly, the SOCS box motif within other proteins is implicated in regulating cytokine secretion (Piessevaux et al., 2008). With the establishment of Rab40b as an important regulator of MMP2/9 targeting to invadopodia, the identification of Rab40b canonical effector proteins, as well as Rab40b-SOCS interacting proteins will be future steps to identifying the mechanisms that regulate MMP2/9 targeting during cancer cell invasion.

CHAPTER III

THE ROLE AND REGULATION OF RAB40B/TKS5 COMPLEX

IN MMP TARGETING AND CANCER CELL INVASION3

Abstract

The prevention of metastasis has posed a major challenge in breast cancer treatment, especially for basal breast cancers. Basal breast cancer is aggressive and metastatic for which limited treatment options are available for patients. Matrix metalloproteinases (MMPs), enzymes that degrade the extracellular matrix, are upregulated in many breast cancers including the basal subtype and therefore have been an attractive therapeutic target for years.

Here, we report that Rab40b is required for MMP2/9 targeting to invadopodia and breast cancer cell invasion in vitro and in vivo. We also demonstrate that Rab40b functions by interacting with Tks5, a known Src kinase substrate and invadopodia-regulating protein.

Significantly, the expression of Rab40b is increased in highly metastatic basal breast tumors, and we demonstrate that Rab40b and Tks5 levels are regulated by miR-204. We show that miR-204 is involved in down-regulating Rab40b and Tks5, thus inhibiting MMP2/9 targeting to invadopodia resulting in a decrease in invadopodia-associated ECM degradation. The study described in this chapter is the first study that identifies and defines a novel

Rab40b/Tks5 and miR-204 dependent invadopodia transport pathway that regulates MMP2/9 secretion and extracellular matrix remodeling during breast cancer progression both in vitro and in vivo.

3This chapter of the thesis is largely based on our article in review Jacob,A., Linklater, E., Lyons, T., Bayless,B., and Prekeris, R. 2015. The role and regulation of Rab40b/Tks5 complex during targeted MMP secretion and breast cancer cell invasion. In Review.

Introduction

Metastasis, or the dissemination of cancer cells from the primary site to secondary sites, is the primary cause for death in cancer patients. The first event that occurs during epithelial cancer metastasis is the breach of the basement membrane (BM), which leads to cell invasion. The BM is a dense and highly specialized extracellular matrix (ECM) that provides structural support to tissues by surrounding all epithelium and endothelium (LeBleu et al., 2007). Breaching the BM is facilitated by actin-rich cellular protrusions known as invadopodia. These invasive matrix-degrading structures were originally identified in cells transformed with activated Src (David-Pfeuty and Singer, 1980; Tarone et al., 1985) and are now known to play an important role during cancer cell invasion in vitro and in vivo.

The matrix degradation activity of invadopodia is attributed to the targeted secretion of matrix-degrading enzymes such as matrix metalloproteinases (MMPs). MMPs are known for their ability to degrade several components of the ECM and are important for normal processes such as tissue remodeling and wound healing. However, aberrant expression and secretion of MMPs has been correlated with promotion of metastasis. In particular, MMP2,

MMP9, and MMP14 have all been shown to promote cancer progression due to their ability to degrade BM components such as Collagen IV. Importantly, MMP2, MMP9 and MMP14 are enriched at the invadopodia (Monsky et al., 1993;Nakahara et al., 1997; Bourguignon et al., 1998; Artym et al., 2006; Clark and Weaver, 2008; Poincloux et al., 2009) and are required for cancer metastasis (Ala-aho and Kahari, 2005; Bjorklund and Koivunen, 2005;

Hofmann et al., 2005; Kerkela and Saarialho-Kere, 2003; Lochter et al., 1998; Mook et al.,

2004; Wagenaar-Miller et al., 2004). Due to the importance of MMPs in cancer progression, much work has been focused on identifying the mechanisms governing targeted MMP

53 secretion. Thus far, it has been shown that accumulation of MMP14 at the invadopodia

(Nakahara et al., 1997; Artym et al., 2006; Clark and Weaver, 2008; Steffen et al., 2008) is regulated by endocytic uptake (Poincloux et al., 2009) and exocytosis (Monteiro et al., 2013) pathways. Additionally, it was shown that MMP14 intracellular traffic is mediated by several endocytic transport proteins such as Rab8 (Bravo-Cordero et al., 2007), the Exocyst complex,

VAMP7 and Tks4 (Buschman et al., 2009).

In contrast to MMP14, the factors governing MMP2 and MMP9 targeting to the invadopodia remain largely unknown. Significantly, it has been shown that MMP2 and

MMP9 are not transported to the invadopodia by endosomes, but instead are targeted directly from the Golgi (Jacob et al., 2013) via microtubule motors like kinesin and by actin regulators like cortactin (Schnaeker et al., 2004, Clark and Weaver, 2008), thus demonstrating that MMP14 and MMP2/9 are targeted to invadopodia via two distinct membrane transport pathways.

Our recent work identified Rab40b GTPase as a protein required for MMP2/9 secretion from the invadopodia in breast cancer cells (Jacob et al., 2013). Additionally, we have also demonstrated that Rab40b is required for cancer cell invasion in vitro (Jacob et al.,

2013). However, how Rab40b regulates MMP2/9 secretion and localized ECM remodeling remains to be understood and the machinery that regulates levels of Rab40b in cancer cells is also unknown. While we have shown that Rab40b is required for MMP2/9 secretion in vitro, it remains unclear whether Rab40b mediates MMP2/9 secretion during breast cancer cell invasion and metastasis in vivo. These questions are the focus of this study. Here we show that Rab40b is required for breast tumor growth and metastasis in vivo and that Rab40b levels are increased in metastatic breast cancers. Since all Rab GTPases function by binding

54 to various regulatory factors, we also screened for Rab40b binding proteins and identified

Tks5 (Tyrosine kinase substrate 5) as a Rab40b binding partner. Importantly, Tks5 is a large scaffolding protein that is phosphorylated by Src kinase and is required for the formation and maturation of invadopodia (Courtneidge et al., 2005b; Sharma et al., 2013). Here, we show that Rab40b is recruited to the invadopodia through its interaction with the Tks5-PX domain and that this binding is likely regulated by Src kinase.

Since, Rab40b is upregulated in metastatic TNBCs (triple negative breast cancers), we also investigated the regulation of Rab40b expression. We demonstrate that miR-204, a known tumor suppressor microRNA, regulates the expression of both Rab40b and Tks5.

While miR-204 has been previously shown to suppress cancer metastasis, the mechanism and the downstream targets that mediate the anti-invasive miR-204 effects remained unclear.

Taken together, this study describes and characterizes a novel Rab40b/Tks5-dependent transport pathway that mediates targeted MMP2/9 secretion during breast cancer metastasis.

Additionally, we show that miR-204 acts as a tumor suppressor by regulating Rab40b and

Tks5 expression and consequently inhibiting MMP2/9 targeting, which leads to a decrease in invadopodia-associated ECM degradation. We propose that this pathway is important for breast tumor metastasis and may represent a novel target for anti-metastatic therapy.

Materials and Methods

Antibodies and constructs

Anti-FLAG and anti-Tubulin antibodies were purchased from Sigma (St Louis, MO).

Alexa-Fluor-594 and Alexa-Fluor-488 conjugated to anti-rabbit and anti-mouse secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).

Alexa-Fluor-568 phalloidin was purchased from Life Technologies (Grand Island, NY).

Collagen I solution was purchased from Corning (Corning, NY). SB3CT (MMP2/9 inhibitor) was purchased from Calbiochem (Billerica, MA). Matrigel was purchased from BD

Biosciences (San Jose, CA). MiR-204 mimic and negative miR control were purchased from

Ambion Life Technologies (Grand Island, NY).

Cell lines

All cell lines were cultured as described previously (Jacob et al. 2013). Briefly,

MDA-MB-231 cells were cultured DMEM with 4.5 g/l glucose, 5.84 g/l L-glutamine and

10% heat-inactivated fetal bovine serum (FBS), and supplemented with 1µg/ml Insulin and

100 IU/ml penicillin and 100 µg/ml streptomycin. MDA-MB-231 cell line stably expressing

FLAG-Rab40b was created by cloning Rab40b into lentiviral pCS2- FLAG vector obtained from Addgene (Cambridge, MA). Stable knockdown and control MDA-MB-231 lines were generated using two Sigma Mission shRNA lentiviral Rab40bshRNA (TRCN0000047529 and TRCN0000047530) plasmids and a non-target shRNA control (SHCLND). All stable cell lines were selected with 1 µg/ml Puromycin and maintained at 370C under a humidified

5% CO2 atmosphere.

GST-Rab40b affinity chromatography

Putative Rab40b binding proteins were identified using a GST-Rab40b affinity column as described previously (Prekeris et al., 2000). Briefly, purified recombinant GST or

GST-Rab40b were coupled to Affi-Gel 10/15 (BioRad, Hercules CA) according to manufacturer’s instructions. Columns were then pre-bound with GMP-PNP. MDA-MB-231 cell Triton X-100 lysate (in 50 mM Hepes, 100 mM NaCl and 5 mM MgCl2) was then applied to both columns, washed with lysate buffer and eluted with 1% SDS. The eluates

56 were then analyzed by the University of Colorado Proteomics Core facility as described previously (Willenborg et al., 2011). Tandem mass spectra were analyzed via Sequest using a human/mouse/rat database concatenated to a randomized human/mouse/rat database.

DTASelect was used to reassemble identified peptides into proteins. Identified proteins were filtered at <5% false discovery rate. Proteins that were identified from GST-Rab40b eluate but were absent in GST only eluate were considered as a candidate Rab40b-binding proteins and are listed in Figure 5A.

GST-Tks5 fragments and glutathione bead pull-down assay

All GST fusion proteins were generated as previously described (Willenborg et al.,

2011). Three domains of Tks5 were cloned into pGEX-2T vector to make GST-Tks5 fragments; PX domain (aa 4-128), SH3-1 domain (aa 129-265), SH3-4 domain (aa 722-936).

Glutathione bead pull downs were performed with 1% Triton X-100 lysates generated from

MDA-MB-231 cells stably expressing FLAG-Rab40b. Lysates were incubated with 10µg of various GST-Tks5 fragments. After incubation, FLAG-Rab40b bound to GST-Tks5 fragments was isolated using glutathione sepharose beads and washed in reaction buffer

(20mM Hepes pH7.4, 150mM NaCl, 1mM MgCl2 and 0.1% Triton X-100). Bound FLAG-

Rab40b was eluted with 1x western running buffer containing 0.2% SDS. Samples were separated by SDS-PAGE and probed with anti-FLAG.

Molecular modeling

All computational modeling studies were conducted with Biovia (formerly Accelrys)

Discovery Studio 4.5 (Biovia Inc., San Diego, CA). Crystal structure coordinates were downloaded from the Protein Data Bank (www.pdb.org). The protein homology model of the human Tks5-PX domain was generated using the MODELLER protocol (Eswar et al., 2008)

57 using the structure of human neutrophil cytosol factor 1 (a.k.a. p47phox PX domain, PDB

ID: 1KQ6; 35% identity, 63% similarity) as a template. The resulting protein structure was subjected to energy minimization utilizing a conjugate gradient minimization with a

CHARMm forcefield (Brooks et al., 2009) and the Generalized Born implicit solvent model with simple switching (Mackerell et al., 2004) until convergence to an RMS gradient of <

0.001 kcal/mol. Figures were generated using Lightwave 2015.2 (NewTek, Inc., San

Antonio, TX).

Inverse invasion assay

Inverse invasion assay with MDA-MB-231 cells was adapted and modified from the version already described in Thun et al. 2011. In brief, a 2.5% gelatin+sucrose and 50 μg/ml fibronectin plug was made on the filter as described in the in situ zymography protocol below. The cells were allowed to invade towards a gradient of 20% FBS and 10% Nu serum for 5 days. The cells were stained with 4µM Calcein for 60 mins and imaged at 10µm steps to a total distance of 180µm with a Leica confocal microscope and LAS AF software.

ImageJ software was used to quantify the number of cells in every 10µm step image from

20µm to 140µm. For quantification, at least 40 cells from 5 different fields per treatment were counted. Data shown are means and standard deviations derived from at least three independent experiments.

In situ zymography

The in situ zymography/matrix degradation assay was done as described previously in

(Jacob et al., 2013). In brief, 18 mm round coverslips in 12-well plates were coated at 37oC with 2.5% gelatin, 2.5% sucrose in PBS. The gelatin was allowed to set at 4°C before cross- linking with 0.5% glutaraldehyde by incubation at 4°C for 15 minutes. A 50 µg/ml solution

58 of FITC-fibronectin (Cytoskeleton, Denver, CO) was then overlaid on top of cross-linked gelatin and incubated in the dark for 1 hour at 4°C. The dish was sterilized with 70% ethanol, washed with DMEM, and equilibrated with invadopodia medium [DMEM supplemented with 20% FBS (Atlanta Biologicals) and 10% Nu-Serum] for 30 minutes. MDA-MB-231 cells (4×104 cells in 2 ml of invadopodia medium) were then added to each well and incubated for 20 hours. The cells were then fixed in 3% paraformaldehyde, permeabilized with 0.4% Triton X-100 in PBS and stained with AlexaFluor-568 phalloidin (Invitrogen,

Carlsbad, CA). To quantify invadopodia formation and localized matrix degradation, ten randomly chosen fields were imaged (using 63× objective) per treatment for each experiment. A total of 260–330 cells were counted in at least three independent experiments.

To measure the number of cells with invadopodia, cells were counted based on the presence of actin puncta and degradation spots seen underneath the cells within the cell boundaries. To measure invadopodia-associated area of degradation, the areas lacking FITC-fibronectin fluorescence were measured using Intelligent Imaging Innovations (Denver, CO) three- dimensional rendering and exploration software. Only degradation areas associated within cell boundaries were analyzed.

RT-PCR and quantitative PCR

Total RNA was extracted from 2×107 MDA-MB-231 cells using TRIzol (Invitrogen) according to the manufacturer's protocol. Reverse transcription to cDNA was performed with iScript Reverse transcription supermix for RT-qPCR. Quantitative PCR was performed using

Taqman PCR master mix from Applied Biosystems (Grand Island, NY). To quantify the efficiency of knockdown, cDNA from mock- or siRNA-treated cells was analyzed in triplicate by qPCR amplification using Taqman Master Mix and Applied Biosystems ViiA7

Real-Time PCR System. The qPCR amplification conditions were: 50°C (2 minutes), 95°C

(10 minutes), 40 cycles at 95°C (15 seconds), 60°C (1 minute). Taqman primers for Rab40b

(Hs00895201_mH) and β-actin (Hs99999903_m1) control were purchased from Applied

Biosystems (Grand Island, NY). Relative quantification was calculated by the ΔΔCT method.

Data is shown as the fold change (averaged from three independent experiments) in Rab40b- knockdown cells compared with mock-treated cells.

Luciferase assay

The Promega Dual Luciferase Reporter Assay protocol and Kit (E1910) was used.

Data was expressed as a ratio of the Renilla luciferase activity to firefly luciferase activity.

The mean and the standard errors have been calculated from three independent experiments.

The luciferase activity was measured using the Turner Biosystems Modulus microplate reader (Madison,WI). For Rab40b, the entire 3’UTR was cloned into the psiCheck-2

Luciferase vector. For Tks5, since the 3’UTR is 8 kb long, two 1 kb pieces containing the miR-204 seed region were cloned adjacent to each other. Mutations in the predicted miR-204 seed regions of the 3’UTR constructs were generated according to the Stratagene

QuikChange site-directed mutagenesis protocol (Santa Clara, CA).

3D Spheroid assay

A 75:25 ratio of Matrigel:CollagenI mixture of 100µl was made into which 12x103 cells were added. For the DQ-gelatin assay, 25g/ml DQ-gelatin (Thermofisher scientific,

Eugene, OR) was added to the Matrigel/Collagen mixture. The cell/Matrigel/Collagen mixture was then spotted onto a 3D chamber slide. The mixture was allowed to harden for 45 mins at 37oC. To each well, 500 ul of 1:1 regular media:growth factor rich media (DMEM +

20% FBS + 10% NuSerum) was added and incubated from 48 hours to 6 days. The spheroids

60 were fixed and stained with anti-Tubulin and AlexaFluor-568 phalloidin antibodies and slides were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Cells were imaged with an inverted Zeiss Axiovert 200M deconvolution microscope with 63x oil immersion lens and Sensicam OE CCD camera. The number and length of invadopodia was quantified from three independent experiments. Image processing was performed using

Intelligent Imaging Innovations (Denver, CO).

Mouse mammary fat pad xenograft assays

Hairless SCID mice (Strain code 474) from Charles River were used for all experiments. Thirty 8 week old female hairless SCID mice were divided into three experimental groups with 10 mice per group. Two million log phase MDA-MB-231 cells

(wild type or stably expressing Rab40b shRNA #1 or Rab40b shRNA #2) were injected into the number four right and left intact mammary glands. Body weight and primary tumor growth was measured weekly using calipers. Once a total tumor burden of 2cm3 was reached, the mice were euthanized. Some mice with ulcerated tumors had to be euthanized earlier than predefined set end point in compliance with the animal research regulations set by the

University of Colorado. Mammary tumors and lungs were harvested. One half of the tissues were flash frozen for qPCR analysis while the other half was fixed and paraffin embedded for histology and immunohistochemical (IH) analysis.

For the time end-point study, twenty 8 week old mice were divided into four experimental groups with 5 mice per group (wild type, shRNA control, Rab40b shRNA#1 and Rab40b shRNA#2). The injections were done as described above and all mice were euthanized at week 8. Mammary tumors and lungs were harvested for qPCR, histology and

IHC analysis.

In situ analysis of lung metastasis

In situ FISH analysis was performed by the University of Colorado Denver

Cytogenetics Core. Briefly, unstained slides with formalin-fixed, paraffin-embedded tissue sections were subjected to a dual-color FISH assay using the Human/Mouse probe set. This probe set was prepared by labeling 1 µg of Human Cot-1 DNA and Mouse Cot-1 DNA

(Invitrogen) respectively with SpectrumRed and SpectrumGreen conjugated dUTPs (Abbott

Molecular) using the Vysis Nick translation kit (Abbott Molecular), according to manufacturer’s instructions. The labeled DNAs were ethanol precipitated with herring sperm

DNA as carrier (1:50) and each pellet was diluted to a final concentration of 50 ng/µl with tDenHybTM-2 hybridization buffer (Insitus Biotechnologies Cat#D102). The probe set was prepared such that it contained 100ng of each DNA per 4.5 µl and this volume was used on every 113mm2 hybridization area. The FFPE specimens were analyzed in an epifluorescence microscope using single interference filters sets for green (FITC), red (Texas red), blue

(DAPI), and dual (red/green) and triple (blue, red, green) band pass. Each section was entirely examined under low magnification and selected areas were investigated under high magnification objectives.

Immunohistochemistry

All paraffin embedded mouse mammary tumors were sectioned for H&E

(Hematoxylin and Eosin) stained. For angiogenesis analysis, tumors isolated 8 weeks after injection from shRNA control and Rab40b KD1 mice were used. Sections 3 and 6 of tumors were stained with anti-CD34 antibody by the University of Colorado Denver Research

Histology Shared Resource. Five random fields from each tumor section were imaged using a

Vert A1 Zeiss microscope and Zen pro 2012 software. Quantification of the area occupied

62 by CD34 stained vessels in each field was performed with ImageJ software. Results are expressed as a ratio of CD34 stained area to total tumor area. Anti-cleaved caspase 3 antibody was used to stain for apoptosis and anti-Ki67 antibody was used to stain for proliferation. The analysis was done as described above for CD34.

Breast cancer data mining

The data shown was obtained from the NCBI GEO dataset GSE58212. The dataset is an mRNA expression profiling of 283 breast cancer samples which was performed using the

SurePrint G3 Human GE 8x60K one-color microarrays from Agilent (Agilent Technologies,

Santa Clara, CA, USA). The data represented in the graph is shown as standard error from the mean and was generated using Prism Graph Pad.

Results

Rab40b is required for breast cancer cell invasion and invadopodia extension

In our previous study, we identified Rab40b as a small monomeric GTPase that is required for MMP2 and MMP9 secretion and invadopodia-associated ECM degradation in

MDA-MB-231 cells cultured on 2D surfaces (Jacob et al., 2013). However, it is becoming widely accepted that 2D invasion and invadopodia formation assays do not always provide accurate information regarding cell invasion machinery. Thus, to define the role of Rab40b in mediating cancer cell invasion through the ECM, we used 3D invasion assays which more closely simulate the in vivo environment (Caswell et al., 2007; Thun et al., 2011). Such 3D invasion assays provide more information as they allow the measurement of the dynamics and invasive capacities of individual cells. To analyze the function of Rab40b in mediating

MMP2/9 secretion in 3D invasion assays, we substituted Matrigel with 2.5% cross-linked

63 gelatin supplemented with 10 µg/ml fibronectin. We chose to use gelatin because it is a known MMP2/9 substrate. Furthermore cross-linked gelatin creates a stiffer 3D matrix (~230

Pa) as compared to a Matrigel matrix (~60-80Pa) (Artym et al., 2015; Van Goethem et al.,

2010). Higher stiffness of extracellular matrix has been shown to induce invadopodia formation and also correlate with poor breast cancer prognosis (Chaudhuri et al., 2014). To test whether Rab40b knockdown affects cell invasion through stiff ECM, we generated

MDA-MB-231 cell lines stably expressing either non-targeting shRNA (control) or two different Rab40b shRNAs, named KD1 (80% Rab40b knockdown) and KD2 (50% Rab40b knockdown) (Fig. 3.1A). Consistent with our previous reports, we found that depletion of

Rab40b decreased MDA-MB-231 cell invasion (Fig. 3.2A). Importantly, treatment of MDA-

MB-231 cells with SB3CT, a known MMP2/9 specific inhibitor, showed comparable decrease in cell invasion ability (Fig. 3.2A), confirming that Rab40b dependent MMP2/9 secretion is required for breast cancer invasion through stiff ECM.

We have previously shown that Rab40b is required for invadopodia-dependent ECM degradation in 2D in situ zymography assays while having no effect on invadopodia formation (Jacob et al., 2013). In 2D assays, cells form multiple invadopodia that lack the physical space to develop into fully mature invasive structures due to the thin layer of matrix plated on a coverslip. Therefore, to determine whether Rab40b has a role in invadopodia formation, maturation and function, we embedded MDA-MB-231 cells stably expressing

FLAG-Rab40b (MDA-MB-231-FLAG-Rab40b) in 9.3 mg/ml Matrigel matrix supplemented with 1mg/ml of Collagen I. Similar to gelatin matrices, Matrigel/Collagen form a stiff matrix

Figure 3.1. Quantification of Rab40b in Rab40b-KD cell lines. (A) Rab40b mRNA levels in wildtype MDA-MB-231 and two different Rab40b shRNA stable cell lines quantified by qPCR. (B) Quantification of Rab40b mRNA levels in SkBr3 and BT549 treated with two different Rab40b siRNAs.

Figure 3.2. Rab40b localizes to the invadopodia and regulates cell invasion in 3D. (A) MDA-MB-231 control, stable Rab40b knockdown (KD1) and SB3CT (MMP2/9 inhibitor) treated cells were plated on a transwell filter containing a gelatin plug and allowed to invade towards a growth factor rich gradient for 5 days. The cells were stained with Calcein and imaged at 10m steps to measure distance of invasion. Data shown are the means and s.e of three independent experiments. (B,E,F) FLAG-Rab40b stable MDA-MB-231 cells were mixed with Matrigel+CollagenI and embedded on a 3D chamber slide and incubated for 48 hrs (B) and 72 hrs (E,F). The spheroids were stained with anti-tubulin and anti-FLAG antibodies. (C,D) MDA-MB-231 cells were embedded in a Matrigel+CollagenI+DQ-gelatin mixture and incubated for 48 hrs.

66 that leads to the formation of MDA-MB-231 spheroids with well-defined invadopodia-like structures. At 48 hours post embedding, many cells already formed well-defined and actin- rich invadopodia precursors or buds (Fig. 3.2B). Even at these early stages of invadopodia formation, some Rab40b accumulation could be observed at the tips of these actin rich structures (Fig. 3.2B). These invadopodia precursors also showed ECM degradation activity as detected by DQ-gelatin around the actin enriched bud (Fig. 3.2C-D). This suggests that while Rab40b is likely not required for initial formation of actin-rich invadopodia precursors,

Rab40b starts accumulating at the tips of invadopodial precursors, thus contributing to invadopodia extension, maturation and ECM degradation. At 4 days post-embedding, the invadopodia precursors extended into long mature invadopodia as marked by the presence of microtubules (Fig. 3.2E and Fig. 3.3A), a known marker for mature invadopodia

(Schoumacher et al., 2010). The tips of these invadopodia-like structures contained actin

(Fig. 3.3A) and were also highly enriched in FLAG-Rab40b (Fig. 3.2F), consistent with the involvement of Rab40b in active transport of vesicles containing cargo, such as MMP2/9, to the invadopodial tips from where it is released for focal degradation of the ECM.

To further test whether Rab40b is required for invadopodia formation and maturation, we next analyzed 4 day spheroids of wild type MDA-MB-231 and cells stably expressing

Rab40b shRNA (KD1 and KD2). As shown in the figure 2B-E, Rab40b knock-down resulted in decreased length of mature invadopodia. Interestingly, while spheroids from both Rab40b knock-down cell lines had shorter invadopodia-like structures (Fig. 3.3E), only KD1 spheroids had decreased numbers of spheroids with invadopodia (Fig. 3.3D). This difference in phenotype severity is likely due to the fact that the KD1 cell line has much better Rab40b knock-down as compared to KD2 (80% and 50% depletion respectively; Fig. 3.1A). After

Figure 3.3. Rab40b is required for invadopodia extension and maturation. (A-E) Wildtype MDA-MB-231 and cells stably expressing Rab40b shRNA (KD1 and KD2) were embedded in Matrigel+CollagenI mixture for 6 days. The spheroids were stained with anti- tubulin and phalloidin antibodies to mark mature invadopodia. Data are represented as means and s.e from three independent experiments.

68 incubating MDA-MB-231 spheroid for 7 days, many of the invadopodia developed into invasive chains containing multiple cells migrating out of the primary spheroid (Fig. 3.4C).

Consistent with the proposed role of Rab40b in regulating cancer metastasis, Rab40b depletion decreased the number of these invasive strands as compared to control MDA-MB-

231 spheroids (Fig. 3.4D). Taken together, the data suggest that Rab40b is primarilyrequired for invadopodia maturation, namely extension and ECM degradation, although we cannot fully rule out a role for Rab40b in the formation or stability of early invadopodia precursors.

Rab40b is required for primary tumor growth and metastasis in vivo

Our in vitro experiments show that Rab40b regulates MMP2/9 targeting to the invadopodia. In order to determine whether Rab40b had any effect on tumor growth and metastasis in vivo, we used SCID mice to perform mammary fat pad injections with either control, KD1 or KD2 MDA-MB-231 cell lines. Surprisingly, eight weeks after tumor cell injection we observed a significant difference in tumor sizes between shRNA control and knockdown mice (Fig. 3.5A). Furthermore, if tumors were allowed to reach a total tumor burden of 2cm3, the rate of primary tumor growth was significantly lower in KD1 or KD2 injected mice (Fig. 3.5B), suggesting that Rab40b plays a role in regulating primary tumor growth. We next sought to explain the decrease in tumor size in Rab40b KD tumors in vivo.

Since Rab40b knock-down does not appear to directly affect cell proliferation in vitro (Fig.

3.6B) we tested whether proliferation and/or apoptosis were altered within the primary tumor. To that end, we analyzed primary tumors harvested eight weeks after injection.

Rab40b knock-down did not cause changes in proliferation or apoptosis in vivo (Fig. 3.7).

Figure 3.4. Rab40b affects the ability of spheroids to form invasive strands. (C) MDA- MB-231 spheroid with a chain of cells migrating out of it after 7 days of incubation in a 3D Matrigel+CollagenI mixture. (D) Quantification showing the number of spheroids with invasive strands in wildtype, shRNA control and Rab40b KD stable cell lines. Data is shown as a percentage of the total number of spheroids counted.

Figure 3.5. Rab40b knockdown affects vascular size and primary tumor growth and metastasis in vivo. (A) Wildtype MDA-MB-231 (Wt), shRNA control (control) and Rab40b knockdown (KD1 and KD2) were implanted in the mammary fat pad of hairless SCID mice. Mice were sacrificed and tumor volumes were measured 8 weeks after injection. n = 10-25 mice per tumor type. Asteriks (*) mark the mice that had to be sacrificed at week 7 due to tumor sizes exceeding guidelines and ulceration. (B) Wildtype (Wt) and Rab40b KD (KD1 and KD2) were implanted in the mammary fat pads of hairless SCID mice. Tumor volumes were measured every week and allowed to grow to a final tumor burden of 2cm3. n = 10-22 per tumor type. (C) Tumors isolated 8 weeks after injection from Control and KD1 mice were analyzed via immunohistochemistry. Vessel area was examined by staining tumor samples with CD34. n = 3 per tumor type. (D) Lungs isolated 8 weeks after injection from Control and KD1 mice were analyzed by dual color FISH assay. n = 3 lungs per tumor group. Scatterplot shows the number of metastases per lung. Metastatic lesions consisting of human cells are indicated as red clumps and marked with white arrows in a sea of green mouse cells and blue DAPI stain. Each image is representative of the entire tumor group.

Figure 3.6. Rab40b depletion affects survival and metastases in mice. (A) Survival curve showing the termination of mice with a total tumor burden of 2cm3 set as the end point. (B) Cell proliferation rates of wildtype MDA-MB-231, shRNA control and Rab40b KD stable cells on tissue culture plastic. Data shown is the mean and s.e from three independent experiments. (C-E) Lungs from shRNA control mice 8 weeks after injection analyzed by dual color FISH assay showing different sizes of metastases.

Figure 3.7. Proliferation and apoptosis in tumors from mice terminated at 8 weeks. (A) Quantification of Ki67 positive areas within each image field for mammary tumors isolated from shRNA control and KD1 mice 8 weeks after injection. (B) Quantification of cleaved caspase 3 positive areas within each image field for mammary tumors isolated from shRNA control and KD1 mice 8 weeks after injection. (C) Representative images showing H&E, Ki67 positive and cleaved caspase 3 positive cells in paraffin embedded mammary tumor sections.

Since, angiogenesis is vital for tumor growth and progression (Weis and Cheresh,

2011) and MMP2/9 have been implicated in angiogenesis (Schnaper et al., 1993; Seftor et al.,

2001) (Itoh et al., 1998b; Vu et al., 1998), we next tested whether Rab40b knock-down is required for primary tumor vascularization. As shown in the figure 3.5C, Rab40b KD tumors had significantly smaller blood vessels compared to the control. Though there were no significant differences in apoptosis at eight weeks after injection, we analyzed whether the decrease in vessel density may cause apoptosis during late tumorigenesis, when tumors are allowed to grow to a final total volume of 2cm3. Consistent with the involvement of Rab40b in mediating transport of MMP2/9 which have been implicated in angiogenesis, there was a significant increase in apoptosis in large KD tumors as compared to control (Fig. 3.8), likely caused by the lack of tumor vascularization, which could lead to hypoxia and eventually apoptosis. Taken together, our results suggest that Rab40b-dependent targeting and secretion of MMP2/9 is a mediator of vasculogenesis to enhance primary tumor growth.

Since our initial studies suggested that Rab40b is required for MDA-MB-231 cell invasion in vitro, we hypothesized that Rab40b knock-down should also affect breast tumor metastasis in vivo. The examination of lungs harvested from control mice 8 weeks after injection, revealed small clumps of disseminated human cells dispersed throughout the lungs

(Fig. 3.5D; human cells in red and marked by arrows). Occasionally, we also observed large lesions in lungs of control mice (Fig. 3.6C-D). In contrast, no large lesions were observed at

8 weeks in mice injected with Rab40b-KD1 cells. Furthermore, the number of lung micro- metastases was significantly decreased (Fig. 3.5D). Thus, our in vivo studies demonstrate that

Rab40b promotes tumor metastasis by regulating primary tumor growth as well as invasion of breast cancer cells.

Figure 3.8. Proliferation and apoptosis in tumors from mice terminated at 2cm3 tumor burden. (A) Quantification of Ki67 positive areas within each image field for mammary tumors isolated after total tumor burden was allowed to grow to approximately 2cm3 from shRNA control and KD1 mice. (B) Quantification of cleaved caspase 3 positive areas within each image field for mammary tumors isolated after total tumor burden was allowed to grow to approximately 2cm3 from shRNA control and KD1 mice. (C) Representative images showing H&E, Ki67 positive and cleaved caspase 3 positive cells in paraffin embedded mammary tumor sections.

Tks5 is a Rab40b binding protein that regulates invadopodia function

All Rab GTPases function by binding to various effector proteins. Thus, identification of Rab40b effector proteins is a key step to understanding the molecular machinery governing Rab40b function. To identify such effector proteins, we incubated MDA-MB-231 cell lysates with either GST or GST-Rab40b-coated beads. Proteins bound to beads were then eluted using 1% SDS and samples were analyzed by mass spectrometry. Only proteins that were identified in GST-Rab40b eluates and not present in GST-only eluates were analyzed further. Additionally, we classified all RNA, DNA or mitochondria associated proteins as contaminants. All the remaining proteins were classified as putative Rab40b-interacting proteins and are listed in figure 3.9A. Interestingly, Tks5 was identified as one of the putative

Rab40b-binding proteins (Fig. 3.9A). Tks5 is a known invadopodia protein that has been reported to regulate invadopodia formation and maturation (Courtneidge et al., 2005a;

Sharma et al., 2013). Furthermore, Tks5 was shown to be required for ECM degradation in vitro (Stylli et al., 2009) as well as tumor growth and metastasis in vivo (Blouw et al., 2015;

Blouw, 2008). As a result, for the remainder of this study, we focused on the role of Tks5 as a Rab40b-binding protein that regulates MMP2/9 targeting and secretion.

To confirm that Tks5 interacts with Rab40b, we immuno-precipitated FLAG-Rab40b from MDA-MB-231 cells stably expressing FLAG-Rab40b (MDA-MB-231-FLAG-Rab40b)

(Fig. 3.9B). Since Rab GTPases cycle between GDP bound inactive and GTP bound active forms, the immunoprecipitation was done in the presence or absence of GTPɣS, a non- hydrolysable form of GTP. Consistent with Tks5 functioning as a Rab40b effector protein,

Tks5 immunoprecipitated with FLAG-Rab40b in the presence of GTPɣS (Fig. 3.9B).

Figure 3.9. Tks5 is a Rab40b binding partner. (A) MDA-MB-231 cell lysates were incubated with GST or GST-Rab40b coated beads and the eluted samples were analyzed by mass spectrometry. The list shows the proteins that were identified in GST-Rab40b eluates and not present in GST. (B) Western blot probed for Tks5 that immuno-precipitation of FLAG-Rab40b from MDA-MB-231 cells stably expressing FLAG-Rab40b in the presence of GTPS. (C) GTP dependent binding of Tks5-PX and FLAG-Rab40b. (D) PX domain homology model showing the position of residues and its hydrophobic interactions in the region of intended mutations. (E) Phospholipid binding assay showing the association of Tks5-PX domain with PI3-P and PI(3,4) P2.

Tks5 is a scaffolding factor that is known to bind to membranes via its PX domain, while interacting with various actin cytoskeleton regulators via its tandem SH3 domains (Fig.

3.10A). In order to identify the Tks5 domain that binds to Rab40b, we generated several

GST-tagged Tks5 fragments, which included the PX and several different SH3 domains (Fig.

3.10 A,B). To test the interaction of these fragments with Rab40b, we then performed glutathione bead pull-down assays using MDA-MB-231-FLAG-Rab40b cell lysates.

Surprisingly, Rab40b did not bind to any of the SH3 domains tested, but rather interacted with Tks5-PX domain (Fig. 3.10B). Furthermore, Tks5-PX and FLAG-Rab40b binding was

GTP-dependent (Fig. 3.9C) confirming that Tks5 is a canonical Rab40b effector protein.

The PX domain is a well-characterized lipid-binding domain that is present in many proteins (Ellson et al., 2002). Typically, PX domains bind to phosphatidyl inositides and are generally considered to mediate a site-specific binding to lipids. Our data suggests that in addition to recruiting Tks5 to the invadopodia precursor at the plasma membrane, the Tks5-

PX domain has a second function of binding Rab40b and thereby tethering/targeting

MMP2/9-transport vesicles to the invadopodia. Structural studies have shown that the PX domains consist of three -sheets and three -helices (Fig. 3.10 C) (Psachoulia and Sansom,

2009). Furthermore, it was shown that the -helices that form the phosphatidyl inositide binding pocket are usually buried within the membrane bilayer (Psachoulia and Sansom,

2009). The only parts of the PX domains that are exposed to the cytosol are the -sheets, suggesting that these regions contain a Rab40b binding motif. Based on this hypothesis, we next analyzed the amino acid sequence of Tks5-PX -sheets looking for a potential Rab40b binding site. We used two main criteria to identify possible Rab40b-binding sites. First, these sites had to be highly conserved among all vertebrates. Second, these sites had to have a

Figure 3.10. Rab40b binds to the PX domain of Tks5. (A) Illustration of Tks5 domains and the different GST-tagged fragments generated for binding assays. (B) Glutathione bead pull down assays with FLAG-Rab40b MDA-MB-231 cell lysates were performed to identify the Tks5 domain that interacts with Rab40b. (C) Illustration of the alpha helices (rectangles), beta sheets (arrows) and the lipid binding region in the PX domain of Tks5. Putative Rab40b binding sites identified from in silico analysis and the mutations generated in these specific sites. (D) Glutathione bead pull down assays with FLAG-Rab40b MDA-MB-231 cell lysates were performed to identify the mutations that can disrupt the binding between Rab40b and Tks5-PX domain. (E-G) The PX domain homology model depicting the position of residues and hydrophobic interactions within the PX domain. (H) Glutathione bead-pull down assays with Y24 and I27 Tks5-PX mutants.

cluster of charged or hydrophobic amino acids that typically mediate protein-protein interactions. This type of in silico analysis led to the identification of two putative Rab40b- binding sites: 14-KRR-19 and 23-YVYI-28 (Fig. 3.10C). Next, we mutated these sites and used glutathione bead pull-down assays to test their binding to FLAG-Rab40b. As shown in figure 3.10D, mutation of 23-YVYI-28 to 23-AVAA-28 disrupted the interaction of FLAG-

Rab40b with Tks5-PX, suggesting that Rab40b bind to the PX domain of Tks5 through this

YVYI motif. In order to further analyze this putative Rab40b binding motif, we next generated a Tks5-PX structure model based on known PX domain structures of other proteins

(Fig. 3.10 E-G). Importantly, with the exception of Y24 and I27, the other residues in the

YVYI motif are buried within the PX domain core and also participate in extensive hydrophobic interactions with other PX domain amino acid residues (Fig. 3.10E-G). Thus it is unlikely these residues can mediate Tks5-PX binding to Rab40b. In contrast, both Y24 and

I27 face to the outside cytosolic face of PX domain and are positioned away from the lipid binding helices and PX-domain core. Therefore, we next mutated Y24 and I27 individually and tested mutant ability to bind Rab40b using glutathione bead pull-down assays. As shown in the figure 3.10H, Y24A, but not I27A, is sufficient to disrupt the interaction between the

PX domain of Tks5 and Rab40b. miR-204 regulates Rab40b and Tks5 expression in breast cancer cells

Thus far, our data has established that Rab40b is required for MMP2/9 targeting to the invadopodia and for breast cancer metastasis in vitro and in vivo. Thus, if Rab40b mediates breast cancer metastasis, Rab40b would be expected to be up-regulated in invasive breast tumors. To test this hypothesis, we analyzed Rab40b expression using publicly available patient breast tumor expression data (NCBI GEO dataset GSE58212). As shown in

80 figure 3.11A, Rab40b is up-regulated in grade 3 breast cancers, but its close homologues

Rab40a and Rab40c are not. Additionally, Rab40b is also highly expressed in basal subtype, which tend to be more metastatic then luminal subtype breast cancers ( Fig. 3.11B). These data are consistent with our previous findings that Rab40b is required for MDA-MB-231 cell invasion (Jacob et al., 2013), since MDA-MB-231 cells are considered basal subtype (Neve et al., 2006).

While our findings demonstrate that Rab40b mediates breast cancer invasion and is upregulated in metastatic tumors, it remains unclear how the expression of Rab40b is controlled. Recently, it was suggested that Rab40b may be regulated by microRNA miR-204

(Li et al., 2011). Importantly, miR-204 is a well-established tumor-suppressor microRNA that is deleted in many cancers and is known to block metastasis (Imam et al., 2012), and miR-204 levels are also decreased in grade 3 metastatic tumors ( Fig. 3.11C). Thus, all these data demonstrate that miR-204 may be an important regulator of cancer cell metastasis. Since it remains unclear how miR-204 affects the cancer invasion machinery, we decided to investigate whether miR-204 inhibits MMP2/9 targeting to invadopodia by decreasing cellular levels of Rab40b. In silico analysis identified a single miR-204 site within the 3’UTR of Rab40b (Fig. 3.12A and Fig. 3.13A). To test whether this site is necessary for miR-204 effect on Rab40b expression, we performed luciferase assays using full-length Rab40b

3’UTR fused to a luciferase reporter. Treatment of cells with a miR-204 mimic resulted in decreased luciferase activity (Fig. 3.12A). In contrast, mutation of the miR-204 seed region blocked the effect of miR-204 mimic on luciferase activity confirming that miR-204 acts by binding to a miR-204 site (216-223) within Rab40b 3’UTR (Fig. 3.12A and Fig. 3.13A). To

Figure 3.11. Rab40b mRNA levels in different grades and subtypes of breast tumors. (A) NCBI GEO dataset GSE58212 analysis showing the upregulation of Rab40b in Grade 3 breast tumors compared to its homologues, Rab40a and Rab40c. (B) Expression of Rab40b and its homologues in highly metastatic basal/triple negative breast cancers compared to other subtypes of breast cancer. (C) Levels of miR-204 in highly metastatic Grade 3 breast tumors. 82

Figure 3.12. miR-204 regulates Rab40b. (A) The 3’UTR of Rab40b contains a single putative miR-204 seed region. The wild type and mutated 3’UTRs of Rab40b were cloned into psiCheck2 dual luciferase reporter vector and cotransfected with miR-204 mimic or negative control into cells. The luciferase activities of Renilla/firefly were analyzed 24 hours after transfection. The data represent the percentage of changes in the ratio of Renilla/firefly activities compared with the controls. The data shown are the means and s.e from 3 independent experiments. (B) Cells were treated with 50nM of miR204 mimic and negative control for 72 hours after which they were harvested and levels of Rab40b mRNA analyzed by qPCR. (C,D) Untreated or Rab40b siRNA-treated MDA-MB-231 cells were plated on gelatin/fibronectin-HiLyte Fluor488 coated coverslips . After 72 hrs of incubation, cells were fixed and stained with rhodamine-phalloidin. (E,F) Quantification of ECM degradation in miR-204 mimic and negative control treated FlagRab40b stably expressing and wildtype MDA-MB-231 cells. Data shown are the means and standard error of three independent experiments. n = 30 per experimental condition. The western blot shows the expression of FLAG-Rab40b in FLAG-Rab40b stable MDA-MB-231 cells.

83 further confirm that Rab40b is a miR-204 target, we treated MDA-MB-231 cells with miR-

204 mimic and analyzed the levels of endogenous Rab40b mRNA by qPCR. As shown in figure 3.12B, miR-204 mimic significantly decreased endogenous Rab40b expression.

Similar results were observed in other breast cancer cell lines tested, such as BT549 and

SkBr3 (Fig. 3.13C).

Since miR-204 decreases cellular levels of Rab40b in breast cancer cells, miR-204 would also be expected to inhibit MMP2/9 targeting to the forming invadopodia. To determine the role of miR-204 regulation of Rab40b levels in invadopodia formation and function, we treated cells with miR-204 mimic and assayed for matrix degradation activity using in situ zymography assays. Reduction of Rab40b levels by miR-204 led to decreased degradation of invadopodia associated extracellular matrix while having no effect on the cells ability to form invadopodia (Fig. 3.12C-D). Overexpression of FLAG-Rab40b in MDA-MB-

231 cells increased ECM degradation (Fig. 3.12E). Importantly, FLAG-Rab40b induced matrix degradation was not affected by miR-204 treatment because the FLAG-Rab40b construct lacks the 3’UTR (Fig. 3.12E). This again demonstrates that miR-204 binds specifically to the Rab40b 3’UTR, thus decreasing cellular levels of Rab40b and thereby regulating invadopodia associated ECM remodeling.

One of the key properties of microRNAs is the ability to regulate multiple target proteins. To test whether miR-204 may affect other invadopodia and MMP2/9 transport regulating proteins, we used in silico analysis (using TargetScan analysis) to search for other putative miR-204 targets. Significantly, we identified two putative miR-204 seed regions

Figure 3.13. MiR-204 seed regions in Tks5 and effects of miR-204 on Rab40b and Tks5 in other breast cancer lines (A) miR-204 seed region in Rab40b 3’UTR. (B) miR-204 seed regions in Tks5 3’UTR. (C) Quantification of Rab40b mRNA levels in miR-204 mimic and negative miR treated SkBr3 and BT549 breast cancer cell lines. (D) Western blot showing Tks5 protein levels in BT549 cells treated with mir-204 mimic or negative miR.

85 within Tks5 3’UTR (Fig. 3.14A and Fig. 3.13B). To test whether any of these miR-204 sites regulate Tks5 levels we again used luciferase assays. Treatment of cells containing the luciferase construct with both miR-204 sites from Tks5 3’UTR led to a decrease in luciferase activity (Fig. 3.14A). Mutating each seed region separately did not abolish the effect of miR-

204 mimic on luciferase activity (Fig. 3.14A, Fig. 3.13B). However, mutating both the first and second seed regions blocked the effect of miR-204 mimic, thus indicating that miR-204 regulates Tk5 by binding to both microRNA sites (Fig. 3.14A). Furthermore, treatment of

MDA-MB-231 or BT549 cell lines with miR-204 mimic resulted in decrease of endogenous

Tks5 (Fig. 3.14B and Fig. 3.13D).

Our aforementioned data shows that Rab40b and Tks5 are both miR-204 targets.

Thus, it is likely that miR-204 inhibits cancer metastasis, at least in part, by blocking invadopodia-dependent ECM degradation and invadopodia extension. To test this hypothesis, we analyzed the effect of miR-204 mimic on invadopodia formation using MDA-MB-231 cells embedded in a Matrigel/Collagen I matrix (Fig. 3.14C-D), also see Fig. 3.2&3.3). While treatment with miR-204 mimic did not affect invadopodia number, it resulted in a significant decrease in invadopodia length (Fig. 3.14C-D). This data is consistent with previous reports showing the requirement of Rab40b and Tks5 in invadopodia formation and maturation, presumably by mediating invadopodia associated ECM degradation (Courtneidge et al.,

2005a; Jacob et al., 2013; Sharma et al., 2013).

Figure 3.14. miR-204 regulates Tks5. (A) The 3’UTR of Tks5 contains 2 putative mir-204 seed regions. Due to the large 8kb size of the Tks5 3’UTR, 1kb fragments containing both the miR-204 seed regions were cloned adjacent to each other into psiCheck2 dual luciferase reporter vector and co-transfected with miR-204 mimic or negative control into cells. (B) Western blot of MDA-MB-231 cells treated with miR-204 mimic and negative control probed with Anti-Tks5 antibody. The graph shows levels of Tks5 as a percentage of mock. Data shown are the means and standard error of three independent experiments. (C-E) MDA- MB-231 cells treated with miR-204 mimic or control mimic were embedded in a Matrigel+CollagenI mixture and incubated for 6days. The spheroids were stained with anti- tubulin and the number and length of invadopodia was quantified. Data shown are the means and standard error of three independent experiments.

Discussion

Even though metastasis is the leading cause of mortality in cancer patients, the cellular and molecular events that occur during this process are not completely understood.

MMPs have been shown to be important for tumor progression and metastasis and therefore have been considered for cancer therapy for many years. One of the obstacles in targeting

MMPs for therapy is that they share a conserved structure. Also, the inhibition of general secretion of these enzymes causes adverse effects, as MMPs are needed for normal processes like wound healing and cell migration. Therefore, it has been necessary to identify molecules involved in specific transport pathways that allow targeted secretion of MMPs. Although

MMP2 and MMP9 have been shown to be upregulated in many cancers and enriched at the invadopodia, there are no defined pathways that describe how these enzymes are transported to specific ECM degradation sites that facilitate metastasis.

We had previously identified Rab40b as a protein required for targeted secretion of

MMP2/9 and invadopodia-associated ECM degradation in vitro (Jacob et al., 2013). Even though in situ zymography and traditional invasion assays allowed us to establish that

Rab40b is involved in MMP2/9 targeting in vitro, the role of Rab40b in relation to invadopodia maturation and ECM degradation in a complex 3D system remained unclear. A

3D matrix provides the physical space for invadopodia maturation and allows cells to invade through a more in vivo like environment. Therefore, in this study, we focused on understanding the function of Rab40b in 3D experimental systems and mouse xenograft models. We have shown that Rab40b is present in early invadopodia precursors suggesting a possible involvement of Rab40b in invadopodia initiation and formation. However, Rab40b is mostly enriched at the tips of mature invadopodia, indicating that Rab40b is important for

88 invadopodia maturation through active targeting of vesicles containing cargo like MMP2/9 to the invadopodial tips, from where MMP2/9 are released for focal ECM degradation.

Degradation of the ECM not only provides space for invadopodia growth, but also results in release/activation of various growth factors required for angiogenesis, tumor growth and metastasis (Kalluri, 2003; Yurchenco, 2011). Thus, Rab40b-dependent MMP2/9 targeting to invadopodia is important in several steps during invadopodia formation and maturation in vitro.

In this study, we also analyzed the role of Rab40b in primary tumor metastasis in vivo using a mouse xenograft model. Surprisingly, we found that Rab40b knock-down resulted in a marked inhibition of primary tumor growth. At least to some extent, the smaller primary tumors after Rab40b knock-down could be caused by aberrant angiogenesis as characterized by smaller blood vessels. Tumors rely heavily on vasculature to expand their growth and metastasis potential. Interestingly, both MMP2 and MMP9 have been implicated in angiogenesis in vitro (Schnaper et al., 1993; Seftor et al., 2001) and in vivo (Itoh et al.,

1998b; Vu et al., 1998). Though the mechanism of how MMP2/9 contributes to angiogenesis remains obscure, the release of VEGF from the ECM due to the degradation activity of these gelatinases is one possible pathway. Upon depletion of Rab40b, the lack of MMP2/9 targeting could lead to reduced angiogenesis, thus inhibiting tumor growth. Consistent with that, in large tumors we could see increased apoptosis, which could be attributed to reduced angiogenesis which will deprive the tumors of nutrients and oxygen leading to cell death.

Another possible explanation for the reduced tumor size in the Rab40b KD mice could be the lack of invasion and dispersal of cells within the tumor. Recently, it was shown that the short-range dispersal or invasion ability of tumor cells within the tumor affects size, shape

89 and growth rate of primary tumors (Waclaw et al., 2015). In our study, we have established that Rab40b is required for invadopodia formation and function. Therefore, in Rab40b KD mice, the cell’s inability to make invadopodia and degrade its surrounding environment could be affecting the movement of cancer cells within the tumor, resulting in smaller tumor size.

The dissemination of cancer cells from the primary tumor is associated with invadopodia formation and extracellular matrix degradation. Our results show significantly smaller number of disseminated human cells in Rab40b KD lungs compared to control.

Furthermore, none of the mice injected with Rab40b-KD cells developed larger lesions, occasionally observed in control mice. However, in this study we were unable to tease apart the direct effect of Rab40b on metastasis since it also affects primary tumor growth. In order to circumvent the effect of Rab40b on tumor size, we analyzed metastasis in lungs from mice with size-matched large tumors over an extended time and found that there was no noted difference in number of disseminated human cells (data not shown). However, while control mice were terminated at about 8 weeks post-injection (due to maximum allowed tumor burden), most Rab40b-KD1 tumors were allowed to grow for 12 weeks to ensure that they reached similar size as controls. Since metastasis depends on tumor size as well as time, it is likely that the additional 4 weeks allowed Rab40b-KD tumors to catch up with their control counterparts with respect to metastasis. Furthermore, qPCR analysis of the tumors harvested after 12 weeks showed that they lost Rab40b shRNA expression (data not shown). Taken together, our data suggest that Rab40b decreases tumor growth and metastasis, potentially as a direct result of cancer cell invasion as well as effects on primary tumor vasculature size.

Another main objective of this study was to identify the mechanisms governing Rab40b function. All Rab GTPases cycle between a GTP bound active form and GDP bound inactive

90 form and function mainly by binding to various effector proteins. Consequently, identification of Rab effector protein(s) is an important step in understanding Rab function and regulation. In order to dissect the mechanism by which Rab40b regulates targeted

MMP2/9 secretion, we used proteomic analysis to identify Tks5 as a Rab40b effector protein

(Fig. 3.15). Importantly, previous reports have established Tks5 as a scaffold protein that is required for invadopodia formation and maturation and regulates tumor growth in vivo

(Blouw, 2008; Courtneidge et al., 2005a). Thus, Tks5 makes an intriguing Rab40b binding partner by mediating Rab40b targeting to the invadopodia plasma membrane to release transport vesicles containing cargo like MMP2 and MMP9.

Tks5 is a large scaffolding protein with a PX domain and five SH3 domains. The PX domain is generally known for its ability to bind to phosphatidylinositol lipids and we and others have shown that the PX domain of Tks5 preferentially associates with PI3P and

PI(3,4)P2 in vitro (Oikawa and Takenawa, 2009; Seals et al., 2005). Additionally, it was suggested that PI(3,4)P2 enriched regions of the plasma membrane recruit Tks5 through its

PX domain, thus initiating invadopodia formation (Sharma et al., 2013). Surprisingly, our data showed that Rab40b also binds to the PX domain of Tks5, suggesting that the Tks5-PX domain plays a dual role of targeting Tks5 to the site of invadopodia formation as well tethering Rab40b transport vesicles (Fig. 3.15). Structural studies of PX domains from several other PX containing proteins have shown that the -helices that form the phosphatidyl inositide binding pocket are usually buried within the membrane bilayer, while the -sheets are exposed to the cytosol (Psachoulia and Sansom, 2009). Based on these data we hypothesized that these -sheets may contain Rab40b-binding motifs. Consistent with this hypothesis, we show that Y24 is located within the second Tks5-PX -sheet and is required

Figure 3.15. Model for Rab40b mediated MMP2/9 targeting to the invadopodia. Tks5 initiates invadopodia formation at PIP2 enriched regions of the plasma membrane and acts as a tethering factor to recruit Rab40b vesicles containing MMP2/9. The interaction between Rab40b and Tks5 allows the release of MMP2/9 in the invadopodia. miR-204 acts as a tumor suppressor by regulating both Rab40b and Tks5 mediated invadopodia formation and function.

92 for Rab40b binding.

Using both in vitro and in vivo models, we demonstrate that Rab40b and its effector protein Tks5, regulate MMP2/9 targeting and secretion during cell invasion and tumor growth (Fig. 3.15). Next, we used publicly available microarray data to analyze Rab40b expression in various breast cancers. Importantly, we discovered that Rab40b mRNA expression was increased in highly metastatic basal breast cancer as well as advanced stages of all breast cancer subtypes. Taken together, these data are consistent with the involvement of Rab40b during breast cancer cell invasion. However, it is unclear how cellular levels of

Rab40b are regulated and why Rab40b expression is increased in metastatic cancers.

Recently, it was suggested that Rab40b may be a miR-204 target (Li et al., 2011). MiR-204 is frequently lost in multiple cancers (Imam et al., 2012) and has been shown to act as a tumor suppressor in gliomas, colorectal and gastric cancers (Liu et al., 2015; Xia et al., 2015; Yin et al., 2014). Furthermore, decreased expression of miR-204 has been reported to correlate with a poor prognosis in breast cancer patients (Li et al., 2014), although how miR-204 suppresses tumor metastasis remains to be understood. Here, we have described a novel mechanism by which miR-204 acts on breast tumorigenesis and metastasis by demonstrating that Rab40b and its binding proteinTks5 are both direct targets of miR-204 (Fig. 3.15). Consistent with this, we also show that miR-204 suppresses invadopodia-associated ECM degradation.

As a whole, our data firmly establish Rab40b as a major regulator of targeted

MMP2/9 secretion and breast tumor growth and metastasis in vivo and in vitro. Furthermore, this study also defines a novel metastasis-regulating pathway that involves Rab40b/Tks5.

Finally, we have shown that cellular levels of both Rab40b and Tks5 are regulated by a known tumor-suppressor microRNA, miR-204. However, many questions remain. It is still

93 unclear whether Rab40b also regulates MMP2/9 targeting in other cancers or whether it is limited to breast cancer. Additionally, while Rab40b regulates targeting of MMP2/9, it is unknown whether Rab40b regulates transport of other MMPs and proteases. At least one of these MMPs, MMP14, is not dependent on Rab40b-vesicles (Jacob et al., 2013), although

MMP2, MMP9 and MMP14 are all enriched at invadopodia (Monsky et al., 1993;Nakahara et al., 1997; Bourguignon et al., 1998; Artym et al., 2006; Clark and Weaver,

2008; Poincloux et al., 2009). While it is clear that MMP2/9 and MMP14 are transported by two different pathways, it is unclear whether these pathways converge at a later stage for efficient delivery of all MMPs to the invadopodia. Interestingly, MMP2 has been shown to be activated by MMP14. Thus, it is possible that MMP2/9 and MMP14 targeting via different transport pathways may function as a “co-incidence detection” system to ensure the fidelity of MMP-dependent ECM remodeling at the invadopodia. Alternatively, different cancers may preferentially use either MMP2/9 or MMP14 targeting pathways.

Rab40b is a unique small monomeric Ras-like GTPase that contains a SOCS box domain, a unique feature attributed only to the Rab40 sub-family of proteins (Stein et al.,

2012). Interestingly, the SOCS box motif in other proteins has been implicated in regulation of cytokine secretion (Piessevaux et al., 2008) and have also been shown to bind

Cullin/Elongin complex, thus activating RING-type E3 ubiquitin ligases and consequently mediating degradation of specific target proteins. Since ubiquitin-dependent protein degradation has emerged as an important modulator of invadopodia and cancer metastasis, the identification of Rab40b-SOCS-interacting proteins might shed more light on the mechanisms that regulate MMP2/9 targeting during cancer cell invasion.

CHAPTER IV

CONCLUSIONS AND FUTURE DIRECTIONS

Conclusions

Despite the fact that MMP2/9 have been shown to be enriched at the invadopodia and are well known for their roles in breast tumor growth and metastasis, prior to this study, very little was known about the machinery mediating MMP2/9 targeting to the invadopodia. Rab

GTPases belong to the Ras superfamily of small GTPases and are known to regulate intracellular vesicle trafficking. There are at least 60 Rabs in humans that are known to be expressed in specific cell types. Previously XRab40, a Xenopus homolog of Rab40 was reported to be involved in Xenopus gastrulation (Lee et al., 2007). However, there is no other description of the regulation and function of Rab40b or the characterization of its binding partners found in the literature.

We have identified Rab40b as an important regulator of MMP2/9 transport and targeted secretion and Tks5, an invadopodia core protein, as a Rab40b binding partner. We have shown that Rab40b interacts with Tks5 through the Y24 residue located in the PX domain of Tks5. Furthermore, we have also shown that both Rab40b and Tks5 are regulated by a known tumor suppressor microRNA, miR-204. Our findings in vitro support the hypothesis that Rab40b regulates MMP2/9 targeting to the invadopodia through its interaction with Tks5 and miR-204 acts as a tumor suppressor by down-regulating both

Rab40b and Tks5, thus inhibiting MMP2/9 targeting to invadopodia resulting in a decrease in invadopodia associated ECM degradation. Thus, our work has identified a novel miR-

204/Rab40b-Tks5-dependent pathway that regulates localized ECM remodelling via

95 targeting MMP2/9 to the forming invadopodia. This is the first study that identifies a highly specific and regulated pathway of MMP2/9 targeting during cancer cell metastasis.

Following our analysis of Rab40b in cultured MDA-MB-231 cells, we sought to determine whether Rab40b regulates MMP2/9 transport in vivo. Our studies were focused on determining the role of Rab40b in primary tumor growth and metastasis. We have shown that

Rab40b is required for primary tumor growth as depletion of Rab40b resulted in slower tumor growth compared to control. Consistent with the implication of MMP2/9 in angiogenesis, we identified decreased vessel size as one of the reasons causing the delay in tumor growth. We observed smaller blood vessels in Rab40b knock downs and increased apoptosis in larger tumors from these mice attributed to deprivation of nutrients and oxygen.

Additionally, we showed that depletion of Rab40b reduced the number of disseminated human cells in the mouse lungs. These findings collectively support our hypothesis that

Rab40b decreases tumor growth and metastasis, potentially as a direct result of cancer cell invasion as well as effects on primary tumor vasculogenesis.

In summary, we have characterized Rab40b in regulating MMP2/9 targeting to the invadopodia both in vitro and in vivo, thus shedding light on a previously unknown topic. We defined a novel miR-204/Rab40b/Tks5 pathway that regulates localized ECM remodeling via targeting MMP2/9 to the invadopodia during breast cancer cell invasion.

Future Directions

Despite the advances our research has provided the field, there is still a lot more to investigate and understand about Rab40b and its mechanism of action. We identified Rab40b as a RabGTPase that regulates secretion of MMP2/9 and showed that it does not regulate

96 transport of MMP14. However, it remains to be identified whether Rab40b is involved in secretion of other MMPs. Although we have shown that Rab40b knock-down has no effect on secretion of cytokines such as IL6 and IL8, it is still unknown what other protein transport pathways are regulated by Rab40b. The identification of more targets would enable us to better characterize Rab40b and its functions in the cell.

All Rabs cycle between GTP bound and GDP bound states. This switch is regulated by guanine nucleotide exchange factors (GEFs) which controls the binding of GTP and

GTPase activating proteins (GAPs) which hydrolyses the GTP to GDP (Grosshans et al.,

2006). The GEFs and GAPs regulate the activity of Rabs temporally and spatially. There still exists a gap in our knowledge of the GEFs and GAPs that enables Rab40b to switch from a

GTP active form to a GDP inactive form and the vesicle tethering proteins utilized to dock

Rab40b positive vesicles on the plasma membrane. This information would enable us to understand the subcellular localization of Rab40b, its site of action and its ability to bind to certain proteins over others. We identified Tks5 as a Rab40b binding partner and showed that this interaction is GTP dependent. However, it still remains to be shown that the interaction between Rab40b and the Tks5-PX domain is required for MMP2/9 secretion from the invadopodia. To answer this question, a rescue experiment with wild type Tks5 and Tks5-PX mutant in a Tks5 knockdown background cell line will be performed. If the wild type Tks5 is able to rescue invadopodia length which is a direct measurement of ECM degradation and the mutant Tks5 does not, then it can be confirmed that the interaction of Rab40b with the Tks5-

PX domain is required for the targeting of MMP2/9 to the invadopodia. Also, it has already been shown that Tks5 is activated and phosphorylated by Src. Therefore, the interesting question that remains to be answered is whether the interaction between Rab40b and Tks5 is

97 dependent on phosphorylation by Src and whether Tks5 phospho mutants or Src kinase dead mutants can inhibit the binding between Rab40b and Tks5.

Another area of interest is the identification of the sequence of events that lead to invadopodia formation. Our finding that Rab40b is present at the invadopodia in its early stages raises the possibility that Rab40b might have a role in invadopodia formation. Further investigation using time lapse imaging with cells stably expressing fluorescently tagged

FLAG- Rab40b and invadopodia markers like Tks5/cortactin or mCherry Lifeact marking filamentous actin would allow us to determine and better define the role of Rab40b and the sequence of events in invadopodia formation. This would enable us to identify specific targets and establish a more concrete mechanism to block invadopodia formation in aggressive cancers. For the purpose of this project, we only pursued Tks5 as Rab40b’s binding partner. However, there are other interesting candidates that were identified from a mass spectrometry analysis done on samples immunoprecipitated with antiFLAG from cell lysates stably expressing FLAG-Rab40b, like SGEF (Src homology 3 domain containing guanine nucleotide exchange factor), TRIM21 (Tripartite motif containing 21) and Elongins

B and C and Cullin5.

SGEF, a RhoG specific guanine nucleotide exchange factor, has been shown to be overexpressed in high grade human glioblastoma tumors and correlates with poor patient survival (Fortin Ensign et al., 2013). SGEF activates RhoG which in turn activates Dock4 which is a GEF for Rac1 (Abraham et al., 2015). Rac1 is postulated to act through several downstream targets to produce free barbed ends on actin filaments resulting in cell migration.

Rac1 stimulates actin polymerization through WAVE proteins, which activates the Arp2/3 complex, which in turn nucleates new actin filaments on the sides of existing filaments

Figure 3.16. Model for Rab40b mediated Src degradation and invadopodia formation through actin regulation. Cullin5 binds to the Cul-box in the SOCS domain of Rab40b and mediates Src degradation and invadopodia disassembly. Data from colorectal cancer patients show a P213L mutation in the Rab40b Cul-box which could prevent the Cullin5 mediated shut down of Src and invadopodia disassembly. In addition to targeting MMPs to the invadopodia, Rab40b regulates invadopodia formation through actin polymerization via the SGEF/RhoG/DOCK4/Rac1 pathway.

99 resulting in the formation of a branched actin filament network (Pollard et al., 2000). In addition to this, Rac1 can also stimulate actin polymerization by generating phophatidylinositol 4,5 bi-phosphate locally, which binds and removes capping proteins on the barbed ends of actin filaments (Carpenter et al., 1999; Tolias et al., 2000). Branched actin assembly is crucial for the formation of invadopodia (Weaver, 2008b). Thus, in addition to regulation of invadopodia maturation and ECM degradation through targeting of MMPs to the invadopodia, Rab40b could also be regulating invadopodia formation through actin polymerization via the SGEF/RhoG/Dock4/Rac1 pathway. Interestingly, it has also been shown that VEGF activates the SGEF/RhoG/DOCK4/Rac1/DOCK9/Cdc42 signalling pathway to control lateral branching and lumen morphogenesis in blood vessels (Abraham et al., 2015). We have attributed the effect of Rab40b KD on vasculature size mice to the implication of MMP2/9 in angiogenesis. However, it is possible that Rab40b could be regulating vasculogenesis through its interaction with SGEF and the pathway that entails.

Therefore, it would be really interesting to analyze the role of Rab40b in vasculogenesis through the SGEF/RhoG/ DOCK4/Rac1/DOCK9/Cdc42 signalling pathway.

The Rab40b sub-family of proteins are different from other Rab GTPases as they contain a SOCS box domain. The SOCS box consists of two core interaction sites, the

BC- box and Cul-box. Elongins B and C bind to the BC-box and function to stabilize the protein which enables the binding of Cullin-5 to the Cul-box (Babon et al., 2008; Linossi and

Nicholson, 2012). Cullins are scaffold proteins for the assembly of Cullin RING domain E3 ubiquitin ligases. TRIM21 is an E3 ubiquitin ligase. It is possible that the SOCS box along with Elongin B and C, Cullin-5 and TRIM21 forms a complex to ubiquitinate specific

100 substrates and target them for degradation. It was recently shown that Cullin-5 represses Src induced tumorigenesis and may act as a tumor suppressor due to its ability to downregulate or inhibit activated Src (Laszlo and Cooper, 2009). Interestingly, our datamining results showed that in colorectal cancer patients, there is a P213L mutation in the Rab40b Cul- box that binds to Cullin-5. Perhaps, this acts as a mechanism to prevent the Cullin-5 mediated shut down of Src and thereby the disassembly of invadopodia associated cancer cell invasion and metastasis. It would be really interesting to do further investigation to determine whether the P213L mutation in the Cul-box of Rab40b affects invadopodia dynamics and function and also stably expressing this mutation in MDA-MB-231 breast cancer cells result in increased tumor growth and metastasis when injected into mice.

Our in vivo studies suggested that Rab40b affects metastasis, but it is still unclear whether Rab40b is important in the earlier or later steps leading to metastasis. The earlier events include the initial breach of the primary tumor basement membrane and intravasation, while the later events include extravasation and establishment of cells in the secondary site.

In order to understand this better, a tail vein injection study needs to be performed. If metastasis is decreased in the Rab40b KD mice similar to that of the mammary fat pad injection study, then it would suggest that Rab40b is important during the later steps of metastasis.

We and others have shown that elevated levels of Rab40b, MMP2 and Tks5 correlate with an increase in metastasis and patient mortality. MiR-204 is a known tumor suppressor that has been shown to inhibit metastasis and is lost in 28% of breast cancers, 40% pediatric renal tumors and 44% of ovarian cancers. An immediate or short term future goal of this project is to investigate the possibility of using Rab40b/miR-204/Tks5 as diagnostic markers.

101

The development of a robust Rab40b antibody would allow the use of Rab40b as a biomarker to screen human tissue samples.

The long term goal of this project is the translational aspect of inhibiting the MMP targeting machinery that may provide a more specific and effective anti-metastasis drug target. Attempts to use generic MMP2/MMP9 inhibitors in clinical trials have proved ineffective, due to non-specific, adverse side effects and pre-existing high levels of secreted

MMP2/9. Thus, targeting the machinery regulating MMP2/9 transport and secretion in breast cancer cells may provide a more specific means of inhibiting breast cancer cell metastasis. A deeper understanding of the molecular properties of the Rab40b/Tks5 interaction will allow us to explore the possibility of using Rab40b/Tks5 complex as a potential and novel therapeutic target. A small molecule screen to identify compounds that can inhibit the

Rab40b/Tks5 interaction could result in the development of a potential drug that might delay primary tumor growth and metastasis in human breast cancer patients. The disruption of the

Rab40b-Tks5-PX interaction is aimed at decreasing invadopodia specific MMP2/9 secretion rather than the general secretion of these MMPs like with MMP inhibitors used in clinical trials previously.

This thesis work has focused on characterizing the role, mechanism and regulation of the Rab40b/Tks5/miR-204 pathway from a cancer aspect. Apart from its role in invadopodia function, cell invasion and primary tumor growth and metastasis, Rab40b has been shown to be important for gastrulation in Xenopus (Lee et al., 2007). Rab40c, a homolog of Rab40b has been shown to be enriched in oligodendrocytes with a possible involvement in myelin formation (Rodriguez-Gabin et al., 2004). Also, neural crest cells in zebrafish were observed to form Src-Tks5 dependent cell protrusions that are required for the migration of these

102 neural crest during embryonic development (Murphy et al., 2011). Therefore, it would be interesting to investigate the role of the Rab40b/Tks5/miR-204 pathway in these normal developmental processes and contribute to understanding the functions of these proteins in detail.

103

REFERENCES

Abraham, S., Scarcia, M., Bagshaw, R.D., McMahon, K., Grant, G., Harvey, T., Yeo, M., Esteves, F.O., Thygesen, H.H., Jones, P.F., et al. (2015). A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis. Nature communications 6, 7286.

Abram, C.L., Seals, D.F., Pass, I., Salinsky, D., Maurer, L., Roth, T.M., and Courtneidge, S.A. (2003). The adaptor protein fish associates with members of the ADAMs family and localizes to podosomes of Src-transformed cells. The Journal of biological chemistry 278, 16844-16851.

Ala-aho, R., and Kahari, V.M. (2005). Collagenases in cancer. Biochimie 87, 273-286.

Artym, V.V., Swatkoski, S., Matsumoto, K., Campbell, C.B., Petrie, R.J., Dimitriadis, E.K., Li, X., Mueller, S.C., Bugge, T.H., Gucek, M., et al. (2015). Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network. The Journal of cell biology 208, 331-350.

Artym, V.V., Zhang, Y., Moiseiwitsch, F.S., Yamada, K.M., and Mueller, S.C. (2006). Dynamic interactions of Cortactin and Membrane Type 1 Matrix Metalloproteinase at Invadopodia:Defining the stages of invadopodia formaton and function. Cancer Research 66, 3034-3043.

Babon, J.J., Sabo, J.K., Soetopo, A., Yao, S., Bailey, M.F., Zhang, J.G., Nicola, N.A., and Norton, R.S. (2008). The SOCS box domain of SOCS3: structure and interaction with the elonginBC-cullin5 ubiquitin ligase. Journal of molecular biology 381, 928-940.

Beaty, B.T., Sharma, V.P., Bravo-Cordero, J.J., Simpson, M.A., Eddy, R.J., Koleske, A.J., and Condeelis, J. (2013). beta1 integrin regulates Arg to promote invadopodial maturation and matrix degradation. Mol Biol Cell 24, 1661-1675, S1661-1611.

Bergers, G., Brekken, R., McMahon, G., Vu, T.H., Itoh, T., Tamaki, K., Tanzawa, K., Thorpe, P., Itohara, S., Werb, Z., et al. (2000). Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nature cell biology 2, 737-744.

Bjorklund, M., and Koivunen, E. (2005). Gelatinase-mediated migration and invasion of cancer cells. Biochimica et biophysica acta 1755, 37-69.

Blouw, B., Patel, M., Iizuka, S., Abdullah, C., You, W.K., Huang, X., Li, J.L., Diaz, B., Stallcup, W.B., and Courtneidge, S.A. (2015). The invadopodia scaffold protein Tks5 is required for the growth of human breast cancer cells in vitro and in vivo. Plos One 10, e0121003.

104

Blouw, B., Seals, D.F., Pass, I., Diaz, B., and Courtneidge, S.A. (2008). A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo. Eur J Cell Biol 87, 555-567.

Blouw, B., Seals, D.F., Pass, I., Diaz, B., Courtneidge, S.A (2008). A role for the podosome/invadopodia scaffold protein Tks5 in tumor growth in vivo. European Journal of Cell Biology 87, 555-567.

Bourguignon, L.Y., Gunja-Smith, Z., and Iida, N. (1998). CD44v(3,8-10) is involved in cytoskelton-mediated tumor cell migration and matrix metalloproteinase MMP-9 association in breast cancer cells. Journal of Cell Physiology 176, 206-215.

Bravo-Cordero, J.J., Marrero-Diaz, R., Megias, D., Genis, L., Garcia-Grande, A., Garcia, M.A., Arroyo, A.G., and Montoya, M.C. (2007). MT1-MMP proinvasive activity is regulated by a novel Rab8-dependent exocytic pathway. The EMBO journal 26, 1499-1510.

Brooks, B.R., Brooks, C.L., 3rd, Mackerell, A.D., Jr., Nilsson, L., Petrella, R.J., Roux, B., Won, Y., Archontis, G., Bartels, C., Boresch, S., et al. (2009). CHARMM: the biomolecular simulation program. Journal of computational chemistry 30, 1545-1614.

Buschman, M.D., Bromann, P.A., Cejudo-Martin, P., Wen, F., Pass, I., and Courtneidge, S.A. (2009). The novel adaptor protein Tks4 (SH3PXD2B) is required for functional podosome formation. Mol Biol Cell 20, 1302-1311.

Busco, G., Cardone, R.A., Greco, M.R., Bellizzi, A., Colella, M., Antelmi, E., Mancini, M.T., Dell'Aquila, M.E., Casavola, V., Paradiso, A., et al. (2010). NHE1 promotes invadopodial ECM proteolysis through acidification of the peri-invadopodial space. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 24, 3903-3915.

Carpenter, C.L., Tolias, K.F., Van Vugt, A., and Hartwig, J. (1999). Lipid kinases are novel effectors of the GTPase Rac1. Advances in enzyme regulation 39, 299-312.

Caswell, P.T., Spence, H.J., Parsons, M., White, D.P., Clark, K., Cheng, K.W., Mills, G.B., Humphries, M.J., Messent, A.J., Anderson, K.I., et al. (2007). Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Developmental cell 13, 496-510.

Chamber, A.F., and Matrisian, L.M. (1997). Changing views of the role of matrix metalloproteinases in metastasis. Journal of the National Cancer Institute 89, 1260-1270.

Chambers, A.F., and Matrisian, L.M. (1997). Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 89, 1260-1270.

105

Chaudhuri, O., Koshy, S.T., Branco da Cunha, C., Shin, J.W., Verbeke, C.S., Allison, K.H., and Mooney, D.J. (2014). Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nature materials 13, 970-978.

Chen, W.T. (1989). Proteolytic activity of specialized surface protrusions formed at rosette contact sites of transformed cells. Journal of Experimental Zoology 251, 167-185 . Chen, W.T., and Wang, J.Y. (1999). Specialized surface protrusions of invasive cells, invadopodia and lamellipodia, have differential MT1-MMP, MMP-2, and TIMP-2 localization. Annals of the New York Academy of Sciences 878, 361-371.

Cheng, J.M., Ding, M., Aribi, A., Shah, P., and Rao, K. (2006). Loss of RAB25 expression in breast cancer. Int J Cancer 118, 2957-2964.

Cheng, K.W., Lahad, J.P., Kuo, W.L., Lapuk, A., Yamada, K., Auersperg, N., Liu, J., Smith- McCune, K., Lu, K.H., Fishman, D., et al. (2004). The RAB25 small GTPase determines aggressiveness of ovarian and breast cancers. Nat Med 10, 1251-1256.

Chuang, Y., Xu, X., Kwiatkowska, A., Tsapraillis, G., Hwang, H., Petritis, K., Flynn, D., and Symons, M. (2012). Regulation of synaptojanin 2 5'-phosphatase activity by Src. Cell Adh Migr 6, 518-525.

Clark, E.S., Brown, B., Whigham, A.S., Kochaishvili, A., Yarbrough, W.G., and Weaver, A.M. (2009). Aggressiveness of HNSCC tumors depends on expression levels of cortactin, a gene in the 11q13 amplicon. Oncogene 28, 431-444.

Clark, E.S., and Weaver, A.M. (2008). A new role for cortactin in invadopodia:regulation of protease secretion. European Journal of Cell Biology 87, 581-590.

Clark, E.S., Whigham, A.S., Yarbrough, W.G., and Weaver, A.M. (2007). Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res 67, 4227-4235.

Coopman, P.J., Do, M.T., Thompson, E.W., and Mueller, S.C. (1998). Phagocytosis of cross- linked gelatin matrix by human breast carcinoma cells correlates with their invasive capacity. Clinical cancer research : an official journal of the American Association for Cancer Research 4, 507-515.

Courtneidge, S.A., Azucena, E.F., Pass, I., Seals, D.F., and Tesfay, L. (2005a). The SRC substrate Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb Symp Quant Biol 70, 167-171.

Courtneidge, S.A., Azucena, E.F., Pass, I., Seals, D.F., and Tesfay, L. (2005b). Src substrate Tks5, podosomes (invadopodia), and cancer cell invasion. Cold Spring Harb Symp Quant Biol 70, 161-171.

106

Coussens, L.M., Tinkle, C.L., Hanahan, D., and Werb, Z. (2000). MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell 103, 481-490.

David-Pfeuty, T., and Singer, S.J. (1980). Altered distributions of the cytoskeletal proteins vinculin and alpha-actinin in cultured fibroblasts transformed by Rous sarcoma virus. Proceedings of the National Academy of Sciences of the United States of America 77, 6687- 6691.

Deryugina, E.I., and Quigley, J.P. (2006). Matrix metalloproteinases and tumor metastasis. Cancer metastasis reviews 25, 9-34.

Edwards, D.R., Handsley, M.M., and Pennington, C.J. (2008). The ADAM metalloproteinases. Molecular aspects of medicine 29, 258-289.

Egeblad, M., and Werb, Z. (2002a). New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer 2, 161-174.

Egeblad, M., and Werb, Z. (2002b). New functions of the matrix metalloproteinases in cancer progression. Nature Reviews Cancer 2, 161-174.

Ellson, C.D., Andrews, S., Stephens, L.R., and Hawkins, P.T. (2002). The PX domain: a new phosphoinositide-binding module. J Cell Sci 115, 1099-1105.

Eswar, N., Eramian, D., Webb, B., Shen, M.Y., and Sali, A. (2008). Protein structure modeling with MODELLER. Methods Mol Biol 426, 145-159.

Fernandez-Catalan, C., Bode, W., Huber, R., Turk, D., Calvete, J.J., Lichte, A., Tschesche, H., and Maskos, K. (1998). Crystal structure of the complex formed by the membrane type 1- matrix metalloproteinase with the tissue inhibitor of metalloproteinases-2, the soluble progelatinase A receptor. The EMBO journal 17, 5238-5248.

Fingleton, B. (2006). Matrix metalloproteinases: roles in cancer and metastasis. Frontiers in bioscience : a journal and virtual library 11, 479-491.

Fortin Ensign, S.P., Mathews, I.T., Eschbacher, J.M., Loftus, J.C., Symons, M.H., and Tran, N.L. (2013). The Src homology 3 domain-containing guanine nucleotide exchange factor is overexpressed in high-grade gliomas and promotes tumor necrosis factor-like weak inducer of apoptosis-fibroblast growth factor-inducible 14-induced cell migration and invasion via tumor necrosis factor receptor-associated factor 2. The Journal of biological chemistry 288, 21887-21897.

Frantz, C., Barreiro, G., Dominguez, L., Chen, X., Eddy, R., Condeelis, J., Kelly, M.J., Jacobson, M.P., and Barber, D.L. (2008). Cofilin is a pH sensor for actin free barbed end formation: role of phosphoinositide binding. The Journal of cell biology 183, 865-879.

107

Gialeli, C., Theocharis, A.D., and Karamanos, N.K. (2011). Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. The FEBS journal 278, 16-27.

Gimona, M., Kaverina, I., Resch, G.P., Vignal, E., and Burgstaller, G. (2003). Calponin repeats regulate actin filament stability and formation of podosomes in smooth muscle cells. Mol Biol Cell 14, 2482-2491.

Gomis-Ruth, F.X., Maskos, K., Betz, M., Bergner, A., Huber, R., Suzuki, K., Yoshida, N., Nagase, H., Brew, K., Bourenkov, G.P., et al. (1997). Mechanism of inhibition of the human matrix metalloproteinase stromelysin-1 by TIMP-1. Nature 389, 77-81.

Gordon, D.E., Bond, L.M., Sahlender, D.A., and Peden, A.A. (2010). A targeted siRNA screen to identify SNAREs required for constitutive secretion in mammalian cells. Traffic 11, 1191-1204.

Greco, M.R., Antelmi, E., Busco, G., Guerra, L., Rubino, R., Casavola, V., Reshkin, S.J., and Cardone, R.A. (2014). Protease activity at invadopodial focal digestive areas is dependent on NHE1-driven acidic pHe. Oncology reports 31, 940-946.

Grosshans, B.L., Ortiz, D., and Novick, P. (2006). Rabs and their effectors: achieving specificity in membrane traffic. Proceedings of the National Academy of Sciences of the United States of America 103, 11821-11827.

Ha, H.Y., Moon, H.B., Nam, M.S., Lee, J.W., Ryoo, Z.Y., Lee, T.H., Lee, K.K., So, B.J., Sato, H., Seiki, M., et al. (2001). Overexpression of membrane-type matrix metalloproteinase-1 gene induces mammary gland abnormalities and adenocarcinoma in transgenic mice. Cancer Res 61, 984-990.

Hagedorn, E.J., Ziel, J.W., Morrissey, M.A., Linden, L.M., Wang, Z., Chi, Q., Johnson, S.A., and Sherwood, D.R. (2013). The netrin receptor DCC focuses invadopodia-driven basement membrane transmigration in vivo. The Journal of cell biology 201, 903-913.

Hiratsuka, S., Nakamura, K., Iwai, S., Murakami, M., Itoh, T., Kijima, H., Shipley, J.M., Senior, R.M., and Shibuya, M. (2002). MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer cell 2, 289-300.

Hofmann, U.B., Houben, R., Brocker, E.B., and Becker, J.C. (2005). Role of matrix metalloproteinases in melanoma cell invasion. Biochimie 87, 307-314.

Hoshino, D., Branch, K.M., and Weaver, A.M. (2013). Signaling inputs to invadopodia and podosomes. J Cell Sci 126, 2979-2989.

Hotary, K., Li, X.Y., Allen, E., Stevens, S.L., and Weiss, S.J. (2006). A cancer cell metalloprotease triad regulates the basement membrane transmigration program. Genes & development 20, 2673-2686.

108

Ikejiri, M., Bernardo, M.M., Bonfil, R.D., Toth, M., Chang, M., Fridman, R., and Mobashery, S. (2005). Potent mechanism-based inhibitors for matrix metalloproteinases. J Biol Chem 280, 33992-34002.

Imam, J.S., Plyler, J.R., Bansal, H., Prajapati, S., Bansal, S., Rebeles, J., Chen, H.I., Chang, Y.F., Panneerdoss, S., Zoghi, B., et al. (2012). Genomic loss of tumor suppressor miRNA- 204 promotes cancer cell migration and invasion by activating AKT/mTOR/Rac1 signaling and actin reorganization. Plos One 7, e52397.

Itoh, T., Tanioka, M., Matsuda, H., Nishimoto, H., Yoshioka, T., Suzuki, R., and Uehira, M. (1999). Experimental metastasis is suppressed in MMP-9-deficient mice. Clinical & experimental metastasis 17, 177-181.

Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T., Nishimoto, H., and Itohara, S. (1998a). Reduced angiogenesis and tumor progression in gelatinase A- deficient mice. Cancer Research 58, 1048-1051.

Itoh, T., Tanioka, M., Yoshida, H., Yoshioka, T., Nishimoto, H., and Itohara, S. (1998b). Reduced angiogenesis and tumor progression in gelatinase A-deficient mice. Cancer Res 58, 1048-1051.

Jacob, A., Jing, J., Lee, J., Schedin, P., Peden, A.A., Junutula, J.R., and Prekeris, R. (2013). Rab40b regulates MMP2 and MMP9 targeting to the invadopodia during breast cancer cell invasion. Journal of Cell Science 126, 4647-4658.

Jiang, A., Lehti, K., Wang, X., Weiss, S.J., Keski-Oja, J., and Pei, D. (2001). Regulation of membrane-type matrix metalloproteinase 1 activity by dynamin-mediated endocytosis. Proceedings of the National Academy of Sciences of the United States of America 98, 13693-13698.

Kakhlon, O., Sakya, P., Larijani, B., Watson, R., and Tooze, S.A. (2006). GGA function is required for maturation of neuroendocrine secretory granules. EMBO J 25, 1590-1602.

Kalluri, R. (2003). Basement membranes: structure, assembly and role in tumour angiogenesis. Nature reviews Cancer 3, 422-433.

Kerkela, E., and Saarialho-Kere, U. (2003). Matrix metalloproteinases in tumor progression: focus on basal and squamous cell skin cancer. Experimental dermatology 12, 109-125.

Kohrmann, A., Kammerer, U., Kapp, M., Dietl, J., and Anacker, J. (2009). Expression of matrix metalloproteinases (MMPs) in primary human breast cancer and breast cancer cell lines: New findings and review of the literature. BMC Cancer 9, 188.

Kruger, A., Fata, J.E., and Khokha, R. (1997). Altered tumor growth and metastasis of a T- cell lymphoma in Timp-1 transgenic mice. Blood 90, 1993-2000.

109

Kruger, A., Sanchez-Sweatman, O.H., Martin, D.C., Fata, J.E., Ho, A.T., Orr, F.W., Ruther, U., and Khokha, R. (1998). Host TIMP-1 overexpression confers resistance to experimental brain metastasis of a fibrosarcoma cell line. Oncogene 16, 2419-2423.

Krzewski, K., Gil-Krzewska, A., Watts, J., Stern, J.N., and Strominger, J.L. (2011). VAMP4- and VAMP7-expressing vesicles are both required for cytotoxic granule exocytosis in NK cells. Eur J Immunol 41, 3323-3329.

Laszlo, G.S., and Cooper, J.A. (2009). Restriction of Src activity by Cullin-5. Current biology : CB 19, 157-162.

Lebeau, A., Nerlich, A.G., Sauer, U., Lichtinghagen, R., and Lohrs, U. (1999). Tissue distribution of major matrix metalloproteinases and their transcripts in human breast carcinomas. Anticancer research 19, 4257-4264.

LeBleu, V.S., Macdonald, B., and Kalluri, R. (2007). Structure and function of basement membranes. Exp Biol Med (Maywood) 232, 1121-1129.

Lee, R.H., Iioka, H., Ohashi, M., Iemura, S., Natsume, T., and Kinoshita, N. (2007). XRab40 and XCullin5 form a ubiquitin ligase complex essential for the noncanonical Wnt pathway. The EMBO journal 26, 3592-3606.

Lehto, V.P., Hovi, T., Vartio, T., Badley, R.A., and Virtanen, I. (1982). Reorganization of cytoskeletal and contractile elements during transition of human monocytes into adherent macrophages. Laboratory investigation; a journal of technical methods and pathology 47, 391-399.

Leong, H.S., Robertson, A.E., Stoletov, K., Leith, S.J., Chin, C.A., Chien, A.E., Hague, M.N., Ablack, A., Carmine-Simmen, K., McPherson, V.A., et al. (2014). Invadopodia are required for cancer cell extravasation and are a therapeutic target for metastasis. Cell reports 8, 1558-1570.

Li, A., Dawson, J.C., Forero-Vargas, M., Spence, H.J., Yu, X., Konig, I., Anderson, K., and Machesky, L.M. (2010). The actin-bundling protein fascin stabilizes actin in invadopodia and potentiates protrusive invasion. Current biology : CB 20, 339-345.

Li, G., Luna, C., Qiu, J., Epstein, D.L., and Gonzalez, P. (2011). Role of miR-204 in the regulation of apoptosis endoplasmic reticulum stress response and inflammation in human trabecular meshwork cells. Biochemistry and Molecular Biology 52, 2999-3007.

Li, W., Jin, X., Zhang, Q., Zhang, G., Deng, X., and Ma, L. (2014). Decreased expression of miR-204 is associated with poor prognosis in patients with breast cancer. International journal of clinical and experimental pathology 7, 3287-3292.

Linder, S., and Kopp, P. (2005). Podosomes at a glance. J Cell Sci 118, 2079-2082.

110

Linder, S., Nelson, D., Weiss, M., and Aepfelbacher, M. (1999). Wiskott-Aldrich syndrome protein regulates podosomes in primary human macrophages. Proceedings of the National Academy of Sciences of the United States of America 96, 9648-9653.

Linder, S., Wiesner, C., and Himmel, M. (2011). Degrading devices: invadosomes in proteolytic cell invasion. Annual review of cell and developmental biology 27, 185-211.

Linossi, E.M., and Nicholson, S.E. (2012). The SOCS box-adapting proteins for ubiquitination and proteasomal degradation. IUBMB life 64, 316-323.

Liu, L., Wang, J., Li, X., Ma, J., Shi, C., Zhu, H., Xi, Q., Zhang, J., Zhao, X., and Gu, M. (2015). MiR-204-5p suppresses cell proliferation by inhibiting IGFBP5 in papillary thyroid carcinoma. Biochemical and biophysical research communications 457, 621-626.

Lizarraga, F., Poincloux, R., Romao, M., Montagnac, G., Le Dez, G., Bonne, I., Rigaill, G., Raposo, G., and Chavrier, P. (2009). Diaphanous-related formins are required for invadopodia formation and invasion of breast tumor cells. Cancer Res 69, 2792-2800.

Lochter, A., Sternlicht, M.D., Werb, Z., and Bissell, M.J. (1998). The significance of matrix metalloproteinases during early stages of tumor progression. Annals of the New York Academy of Sciences 857, 180-193.

Lohmer, L.L., Kelley, L.C., Hagedorn, E.J., and Sherwood, D.R. (2014). Invadopodia and basement membrane invasion in vivo. Cell Adh Migr 8, 246-255.

Mackerell, A.D., Jr., Feig, M., and Brooks, C.L., 3rd (2004). Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. Journal of computational chemistry 25, 1400-1415.

Mader, C.C., Oser, M., Magalhaes, M.A., Bravo-Cordero, J.J., Condeelis, J., Koleske, A.J., and Gil-Henn, H. (2011). An EGFR-Src-Arg-cortactin pathway mediates functional maturation of invadopodia and breast cancer cell invasion. Cancer Res 71, 1730-1741.

Marchisio, P.C., Cirillo, D., Naldini, L., Primavera, M.V., Teti, A., and Zambonin-Zallone, A. (1984). Cell-substratum interaction of cultured avian osteoclasts is mediated by specific adhesion structures. The Journal of cell biology 99, 1696-1705.

Mehner, C., Hockla, A., Miller, E., Ran, S., Radisky, D.C., and Radisky, E.S. (2014). Tumor cell-produced matrix metalloproteinase 9 (MMP-9) drives malignant progression and metastasis of basal-like triple negative breast cancer. Oncotarget 5, 2736-2749.

Monsky, W.L., Kelly, T., and Lin, C.Y. (1993). Binding and localization of M(r) 72,000 matrix metalloproteinase at cell surface invadopodia. Cancer Research 53, 3159-3164.

111

Monteiro, P., Rosse, C., Castro-Castro, A., Irondelle, M., Lagoutte, E., Paul-Gilloteaux, P., Desnos, C., Formstecher, E., Darchen, F., Perrais, D., et al. (2013). Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia. The Journal of cell biology 203, 1063-1079.

Mook, O.R., Frederiks, W.M., and Van Noorden, C.J. (2004). The role of gelatinases in colorectal cancer progression and metastasis. Biochimica et biophysica acta 1705, 69-89.

Moreau, V., Tatin, F., Varon, C., and Genot, E. (2003). Actin can reorganize into podosomes in aortic endothelial cells, a process controlled by Cdc42 and RhoA. Molecular and cellular biology 23, 6809-6822.

Mori, H., Lo, A.T., Inman, J.L., Alcaraz, J., Ghajar, C.M., Mott, J.D., Nelson, C.M., Chen, C.S., Zhang, H., Bascom, J.L., et al. (2013). Transmembrane/cytoplasmic, rather than catalytic, domains of Mmp14 signal to MAPK activation and mammary branching morphogenesis via binding to integrin beta1. Development 140, 343-352.

Mueller, S.C., Ghersi, G., Akiyama, S.K., Sang, Q.X., Howard, L., Pineiro-Sanchez, M., Nakahara, H., Yeh, Y., and Chen, W.T. (1999). A novel protease-docking function of integrin at invadopodia. The Journal of biological chemistry 274, 24947-24952.

Murphy, D.A., and Courtneidge, S.A. (2011a). The 'ins' and 'outs' of podosomes and invadopodia: characteristics, formation and function. Nat Rev Mol Cell Biol 12, 413-426.

Murphy, D.A., and Courtneidge, S.A. (2011b). The 'ins' and 'outs' of podosomes and invadopodia:characteristics, formation and function. Nature Reviews Molecular Cell Biology 12, 413-426.

Murphy, D.A., Diaz, B., Bromann, P.A., Tsai, J.H., Kawakami, Y., Maurer, J., Stewart, R.A., Izpisua-Belmonte, J.C., and Courtneidge, S.A. (2011). A Src-Tks5 pathway is required for neural crest cell migration during embryonic development. Plos One 6, e22499.

Murphy, G., and Crabbe, T. (1995). Gelatinases A and B. Methods in enzymology 248, 470- 484.

Murphy, G., and Gavrilovic, J. (1999). Proteolysis and cell migration: creating a path? Curr Opin Cell Biol 11, 614-621.

Nakahara, H., Howard, L., Thompson, E.W., Sato, H., Seiki, M., Yeh, Y., and Chen, W.T. (1997). Transmembrane/cytoplasmic domain-mediated membrane type1-matrix metalloprotease docking to invadopodiais required for cell invasion. PNAS 94, 7959-7964.

Neve, R.M., Chin, K., Fridlyand, J., Yeh, J., Baehner, F.L., Fevr, T., Clark, L., Bayani, N., Coppe, J.P., Tong, F., et al. (2006). A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer cell 10, 515-527.

112

Oikawa, T., and Takenawa, T. (2009). PtdIns (3,4) P2 instigates focal adhesions to generate podosomes. Cell Adh Migr 3, 195-197.

Oser, M., Yamaguchi, H., Mader, C.C., Bravo-Cordero, J.J., Arias, M., Chen, X., Desmarais, V., van Rheenen, J., Koleske, A.J., and Condeelis, J. (2009). Cortactin regulates cofilin and N-WASp activities to control the stages of invadopodium assembly and maturation. The Journal of cell biology 186, 571-587.

Osiak, A.E., Zenner, G., and Linder, S. (2005). Subconfluent endothelial cells form podosomes downstream of cytokine and RhoGTPase signaling. Exp Cell Res 307, 342-353.

Overall, C.M., and Kleifeld, O. (2006). Towards third generation matrix metalloproteinase inhibitors for cancer therapy. Br J Cancer 94, 941-946.

Pacheco, M.M., Mourao, M., Mantovani, E.B., Nishimoto, I.N., and Brentani, M.M. (1998). Expression of gelatinases A and B, stromelysin-3 and matrilysin genes in breast carcinomas: clinico-pathological correlations. Clinical & experimental metastasis 16, 577-585.

Peden, A.A., Schonteich, E., Chun, J., Junutula, J.R., Scheller, R.H., and Prekeris, R. (2004). The RCP-Rab11 complex regulates endocytic protein sorting. Mol Biol Cell 15, 3530-3541.

Pfeffer, S.R. (2001). Rab GTPases: specifying and deciphering organelle identity and function. Trends Cell Biol 11, 487-491.

Piessevaux, J., Lavens, D., Peelman, F., and Tavernier, J. (2008). The many faces of the SOCS box. Cytokine Growth Factor Rev 19, 371-381.

Poincloux, R., Lizarraga, F., and Chavrier, P. (2009a). Matrix invasion by tumor cells: a focus on MT1-MMP trafficking to invadopodia. Journal of Cell Science 122, 3015-3024.

Poincloux, R., Lizarraga, F., and Chavrier, P. (2009b). Matrix invasion by tumour cells: a focus on MT1-MMP trafficking to invadopodia. J Cell Sci 122, 3015-3024.

Polette, M., Nawrocki-Raby, B., Gilles, C., Clavel, C., and Birembaut, P. (2004). Tumour invasion and matrix metalloproteinases. Critical reviews in oncology/hematology 49, 179- 186. Pollard, T.D., Blanchoin, L., and Mullins, R.D. (2000). Molecular mechanisms controlling actin filament dynamics in nonmuscle cells. Annual review of biophysics and biomolecular structure 29, 545-576.

Pope, B.J., Zierler-Gould, K.M., Kuhne, R., Weeds, A.G., and Ball, L.J. (2004). Solution structure of human cofilin: actin binding, pH sensitivity, and relationship to actin- depolymerizing factor. The Journal of biological chemistry 279, 4840-4848.

Prekeris, R., Klumperman, J., and Scheller, R.H. (2000). A Rab11/Rip11 protein complex regulates apical membrane trafficking via recycling endosomes. Molecular cell 6, 1437-1448.

113

Psachoulia, E., and Sansom, M.S. (2009). PX- and FYVE-mediated interactions with membranes: simulation studies. Biochemistry 48, 5090-5095.

Quintavalle, M., Elia, L., Condorelli, G., and Courtneidge, S.A. (2010). MicroRNA control of podosome formation in vascular smooth muscle cells in vivo and in vitro. J Cell Biol 189, 13-22.

Remacle, A., Murphy, G., and Roghi, C. (2003). Membrane type I-matrix metalloproteinase (MT1-MMP) is internalised by two different pathways and is recycled to the cell surface. J Cell Sci 116, 3905-3916.

Rodriguez-Gabin, A.G., Almazan, G., and Larocca, J.N. (2004). Vesicle transport in oligodendrocytes: probable role of Rab40c protein. Journal of neuroscience research 76, 758- 770.

Roskelley, C.D., and Bissell, M.J. (1995). Dynamic reciprocity revisited: a continuous, bidirectional flow of information between cells and the extracellular matrix regulates mammary epithelial cell function. Biochem Cell Biol 73, 391-397.

Roskelley, C.D., Srebrow, A., and Bissell, M.J. (1995). A hierarchy of ECM-mediated signalling regulates tissue-specific gene expression. Curr Opin Cell Biol 7, 736-747.

Sakamoto, T., and Seiki, M. (2010). A membrane protease regulates energy production in macrophages by activating hypoxia-inducible factor-1 via a non-proteolytic mechanism. The Journal of biological chemistry 285, 29951-29964.

Sakurai-Yageta, M., Recchi, C., Le Dez, G., Sibarita, J.B., Daviet, L., Camonis, J., D'Souza- Schorey, C., and Chavrier, P. (2008). The interaction of IQGAP1 with the exocyst complex is required for tumor cell invasion downstream of Cdc42 and RhoA. The Journal of cell biology 181, 985-998.

Sameni, M., Dosescu, J., Yamada, K.M., Sloane, B.F., and Cavallo-Medved, D. (2008). Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells. Molecular imaging 7, 199-213.

Sang, Q.A., Bodden, M.K., and Windsor, L.J. (1996). Activation of human progelatinase A by collagenase and matrilysin: activation of procollagenase by matrilysin. Journal of protein chemistry 15, 243-253.

Schmalfeldt, B., Prechtel, D., Harting, K., Spathe, K., Rutke, S., Konik, E., Fridman, R., Berger, U., Schmitt, M., Kuhn, W., et al. (2001). Increased expression of matrix metalloproteinases (MMP)-2, MMP-9, and the urokinase-type plasminogen activator is associated with progression from benign to advanced ovarian cancer. Clin Cancer Res 7, 2396-2404.

114

Schnaeker, E.M., Ossig, R., Ludwig, T., Dreier, R., Oberleithner, H., Wilhelmi, M., and Schneider, S.W. (2004). Microtubule-dependent matrix metalloproteinase-2/matrix metalloproteinase-9 exocytosis: prerequisite in human melanoma cell invasion. Cancer Res 64, 8924-8931.

Schnaper, H.W., Grant, D.S., Stetler-Stevenson, W.G., Fridman, R., D'Orazi, G., Murphy, A.N., Bird, R.E., Hoythya, M., Fuerst, T.R., French, D.L., et al. (1993). Type IV collagenase(s) and TIMPs modulate endothelial cell morphogenesis in vitro. Journal of cellular physiology 156, 235-246.

Schoumacher, M., Goldman, R.D., Louvard, D., and Vignjevic, D.M. (2010). Actin, microtubules, and vimentin intermediate filaments cooperate for elongation of invadopodia. The Journal of cell biology 189, 541-556.

Scorilas, A., Karameris, A., Arnogiannaki, N., Ardavanis, A., Bassilopoulos, P., Trangas, T., and Talieri, M. (2001). Overexpression of matrix-metalloproteinase-9 in human breast cancer: a potential favourable indicator in node-negative patients. British journal of cancer 84, 1488-1496.

Seals, D.F., Azucena, E.F., Jr., Pass, I., Tesfay, L., Gordon, R., Woodrow, M., Resau, J.H., and Courtneidge, S.A. (2005). The adaptor protein Tks5/Fish is required for podosome formation and function, and for the protease-driven invasion of cancer cells. Cancer cell 7, 155-165.

Seftor, R.E., Seftor, E.A., Koshikawa, N., Meltzer, P.S., Gardner, L.M., Bilban, M., Stetler- Stevenson, W.G., Quaranta, V., and Hendrix, M.J. (2001). Cooperative interactions of laminin 5 gamma2 chain, matrix metalloproteinase-2, and membrane type-1- matrix/metalloproteinase are required for mimicry of embryonic vasculogenesis by aggressive melanoma. Cancer Res 61, 6322-6327.

Seiler, C., Davuluri, G., Abrams, J., Byfield, F.J., Janmey, P.A., and Pack, M. (2012). Smooth muscle tension induces invasive remodeling of the zebrafish intestine. PLoS biology 10, e1001386.

Shah, F.D., Shukla, S.N., Shah, P.M., Shukla, H.K., and Patel, P.S. (2009). Clinical significance of matrix metalloproteinase 2 and 9 in breast cancer. Indian J Cancer 46, 194- 202.

Sharma, V.P., Eddy, R., Entenberg, D., Kai, M., Gertler, F.B., and Condeelis, J. (2013). Tks5 and SHIP2 regulate invadopodium maturation, but not initiation, in breast carcinoma cells. Current biology : CB 23, 2079-2089.

Soloway, P.D., Alexander, C.M., Werb, Z., and Jaenisch, R. (1996). Targeted mutagenesis of Timp-1 reveals that lung tumor invasion is influenced by Timp-1 genotype of the tumor but not by that of the host. Oncogene 13, 2307-2314.

115

Steegmaier, M., Klumperman, J., Foletti, D.L., Yoo, J.S., and Scheller, R.H. (1999). Vesicle- associated membrane protein 4 is implicated in trans-Golgi network vesicle trafficking. Mol Biol Cell 10, 1957-1972.

Steffen, A., Le Dez, G., Poincloux, R., Recchi, C., Nassoy, P., Rottner, K., Galli, T., and Chavrier, P. (2008). MT1-MMP-dependent invasion is regulated by TI-VAMP/VAMP7. Current biology : CB 18, 926-931.

Stein, M., Pilli, M., Bernauer, S., Habermann, B.H., Zerial, M., and Wade, R.C. (2012). The interaction properties of the human Rab GTPase family--comparative analysis reveals determinants of molecular binding selectivity. Plos One 7, e34870.

Sternlicht, M.D., Lochter, A., Sympson, C.J., Huey, B., Rougier, J.P., Gray, J.W., Pinkel, D., Bissell, M.J., and Werb, Z. (1999). The stromal proteinase MMP3/stromelysin-1 promotes mammary carcinogenesis. Cell 98, 137-146.

Sternlicht, M.D., and Werb, Z. (2001). How matrix metalloproteinases regulate cell behavior. Annual review of cell and developmental biology 17, 463-516.

Strongin, A.Y., Collier, I., Bannikov, G., Marmer, B.L., Grant, G.A., and Goldberg, G.I. (1995). Mechanism of cell surface activation of 72-kDa type IV collagenase. Isolation of the activated form of the membrane metalloprotease. The Journal of biological chemistry 270, 5331-5338.

Stylli, S.S., Kaye, A.H., and Lock, P. (2008). Invadopodia: at the cutting edge of tumour invasion. Journal of clinical neuroscience : official journal of the Neurosurgical Society of Australasia 15, 725-737.

Stylli, S.S., Stacey, T.T., Verhagen, A.M., Xu, S.S., Pass, I., Courtneidge, S.A., and Lock, P. (2009). Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation. J Cell Sci 122, 2727-2740.

Sun, J., Lu, F., He, H., Shen, J., Messina, J., Mathew, R., Wang, D., Sarnaik, A.A., Chang, W.C., Kim, M., et al. (2014). STIM1- and Orai1-mediated Ca2+ oscillation orchestrates invadopodium formation and melanoma invasion. The Journal of cell biology 207, 535-548.

Sutoh, M., Hashimoto, Y., Yoneyama, T., Yamamoto, H., Hatakeyama, S., Koie, T., Okamoto, A., Yamaya, K., Saitoh, H., Funyu, T., et al. (2010). Invadopodia formation by bladder tumor cells. Oncology research 19, 85-92.

Takeha, S., Fujiyama, Y., Bamba, T., Sorsa, T., Nagura, H., and Ohtani, H. (1997). Stromal expression of MMP-9 and urokinase receptor is inversely associated with liver metastasis and with infiltrating growth in human colorectal cancer: a novel approach from immune/inflammatory aspect. Japanese journal of cancer research : Gann 88, 72-81.

116

Takkunen, M., Hukkanen, M., Liljestrom, M., Grenman, R., and Virtanen, I. (2010). Podosome-like structures of non-invasive carcinoma cells are replaced in epithelial- mesenchymal transition by actin comet-embedded invadopodia. Journal of cellular and molecular medicine 14, 1569-1593.

Tarone, G., Cirillo, D., Giancotti, F.G., Comoglio, P.M., and Marchisio, P.C. (1985). Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes. Exp Cell Res 159, 141-157.

Thun, A., Birtwistle, M., Kalna, G., Grindlay, J., Strachan, D., Kolch, W., von Kriegsheim, A., and Norman, J.C. (2011). ERK2 drives tumor cell migration in thre-dimensional micorenvironments by suppressing expression of Rab17 and liprin. Journal of Cell Science 125, 1465-1477.

Tolde, O., Rosel, D., Vesely, P., and Brabek, J. (2010). The structure of invadopodia in a complex 3D environment. European Journal of Cell Biology 89, 674-680.

Tolias, K.F., Hartwig, J.H., Ishihara, H., Shibasaki, Y., Cantley, L.C., and Carpenter, C.L. (2000). Type Ialpha phosphatidylinositol-4-phosphate 5-kinase mediates Rac-dependent actin assembly. Current biology : CB 10, 153-156. van 't Veer, L.J., Dai, H., van de Vijver, M.J., He, Y.D., Hart, A.A., Mao, M., Peterse, H.L., van der Kooy, K., Marton, M.J., Witteveen, A.T., et al. (2002). Gene expression profiling predicts clinical outcome of breast cancer. Nature 415, 530-536. van den Oord, J.J., Paemen, L., Opdenakker, G., and de Wolf-Peeters, C. (1997). Expression of gelatinase B and the extracellular matrix metalloproteinase inducer EMMPRIN in benign and malignant pigment cell lesions of the skin. The American journal of pathology 151, 665- 670.

Van Goethem, E., Poincloux, R., Gauffre, F., Maridonneau-Parini, I., and Le Cabec, V. (2010). Matrix architecture dictates three-dimensional migration modes of human macrophages: differential involvement of proteases and podosome-like structures. J Immunol 184, 1049-1061.

Visse, R., and Nagase, H. (2003). Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res 92, 827-839.

Vu, T.H., Shipley, J.M., Bergers, G., Berger, J.E., Helms, J.A., Hanahan, D., Shapiro, S.D., Senior, R.M., and Werb, Z. (1998). MMP-9/gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411-422.

Waclaw, B., Bozic, I., Pittman, M.E., Hruban, R.H., Vogelstein, B., and Nowak, M.A. (2015). A spatial model predicts that dispersal and cell turnover limit intratumour heterogeneity. Nature 525, 261-264.

117

Wagenaar-Miller, R.A., Gorden, L., and Matrisian, L.M. (2004). Matrix metalloproteinases in colorectal cancer: is it worth talking about? Cancer metastasis reviews 23, 119-135.

Weaver, A.M. (2008a). Cortactin in tumor invasiveness. Cancer Lett 265, 157-166.

Weaver, A.M. (2008b). Invadopodia. Current biology : CB 18, R362-364.

Weis, S.M., and Cheresh, D.A. (2011). Tumor angiogenesis: molecular pathways and therapeutic targets. Nature medicine 17, 1359-1370.

Willenborg, C., Jing, J., Wu, C., Matern, H., Schaack, J., Burden, J., and Prekeris, R. (2011). Interaction between FIP5 and SNX18 regulates epithelial lumen formation. The Journal of cell biology 195, 71-86.

Woessner, J.F., Jr. (1991). Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB journal : official publication of the Federation of American Societies for Experimental Biology 5, 2145-2154.

Wolf, K., Wu, Y.I., Liu, Y., Geiger, J., Tam, E., Overall, C., Stack, M.S., and Friedl, P. (2007). Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nature cell biology 9, 893-904.

Xia, Z., Liu, F., Zhang, J., and Liu, L. (2015). Decreased Expression of MiRNA-204-5p Contributes to Glioma Progression and Promotes Glioma Cell Growth, Migration and Invasion. Plos One 10, e0132399.

Yamaguchi, H., Lorenz, M., Kempiak, S., Sarmiento, C., Coniglio, S., Symons, M., Segall, J., Eddy, R., Miki, H., Takenawa, T., et al. (2005a). Molecular mechanisms of invadopodium formation: the role of the N-WASP-Arp2/3 complex pathway and cofilin. The Journal of cell biology 168, 441-452.

Yamaguchi, H., Wyckoff, J., and Condeelis, J. (2005b). Cell migration in tumors. Current Opinion in Cell Biology 17, 559-564.

Yamaguchi, H., Yoshida, S., Muroi, E., Yoshida, N., Kawamura, M., Kouchi, Z., Nakamura, Y., Sakai, R., and Fukami, K. (2011). Phosphoinositide 3-kinase signaling pathway mediated by p110alpha regulates invadopodia formation. The Journal of cell biology 193, 1275-1288.

Yamamoto, H., Sutoh, M., Hatakeyama, S., Hashimoto, Y., Yoneyama, T., Koie, T., Saitoh, H., Yamaya, K., Funyu, T., Nakamura, T., et al. (2011). Requirement for FBP17 in invadopodia formation by invasive bladder tumor cells. The Journal of urology 185, 1930- 1938.

Yang, W., Arii, S., Gorrin-Rivas, M.J., Mori, A., Onodera, H., and Imamura, M. (2001). Human macrophage metalloelastase gene expression in colorectal carcinoma and its clinicopathologic significance. Cancer 91, 1277-1283.

118

Yin, Y., Zhang, B., Wang, W., Fei, B., Quan, C., Zhang, J., Song, M., Bian, Z., Wang, Q., Ni, S., et al. (2014). miR-204-5p inhibits proliferation and invasion and enhances chemotherapeutic sensitivity of colorectal cancer cells by downregulating RAB22A. Clinical cancer research : an official journal of the American Association for Cancer Research 20, 6187-6199.

Yurchenco, P.D. (2011). Basement membranes: cell scaffoldings and signaling platforms. Cold Spring Harbor perspectives in biology 3.

Zeng, Z.S., Huang, Y., Cohen, A.M., and Guillem, J.G. (1996). Prediction of colorectal cancer relapse and survival via tissue RNA levels of matrix metalloproteinase-9. Journal of clinical oncology : official journal of the American Society of Clinical Oncology 14, 3133- 3140.

119