Regulation of Breast Cancer Cell Proliferation by APRIL

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2016 Regulation of Breast Cancer Cell Proliferation by APRIL

Matook, Wejdan

Matook, W. (2016). Regulation of Breast Cancer Cell Proliferation by APRIL (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/27583 http://hdl.handle.net/11023/3081 master thesis

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UNIVERSITY OF CALGARY

Regulation of Breast Cancer Cell Proliferation by APRIL

Wejdan Matook

A THESIS

SUBMITTED TO THE FACULTY OF GRADUATE STUDIES

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE

DAGREE OF MASTER OF SCIENCE

GRADUATE PROGRAM IN MEDICAL SCIENCE

CALGARY, ALBERTA

June, 2016

Abstract

Breast cancer is the most common cause of death from cancer among women. “A proliferation- inducing ligand” (APRIL) is seen in the stroma of approximately 38% of breast cancer patients.

APRIL is a tumor necrosis factor superfamily member that is implicated in lymphoid cell survival, proliferation and apoptosis. APRIL studies initially focused on lymphoid cells as known APRIL receptors are exclusive to these cells. However, I found that APRIL promotes breast cancer cell proliferation, and an APRIL-specific blocking peptide inhibits APRIL-induced proliferation.

Therefore, I sought to identify APRIL targets in breast cancer cells. Among those identified, colony-stimulating factor 2 receptor beta (CSF2RB) is interesting. CSF2RB-APRIL interaction is direct, and the CSF2RB ligand, CSF2, has 41% amino acid sequence similarity to APRIL.

CSF2RB-linked Akt and STAT3 signaling are activated in APRIL-mediated breast cancer cell proliferation. Thus, my findings raise the possibility that CSF2RB is a novel APRIL receptor in non-lymphoid cells.

Acknowledgements

I would like to thank both Drs. Ki-Young Lee and Susie Rosales for providing me the opportunity to conduct a very exciting project for my M.Sc. degree at the University of Calgary. I thank both

Drs. Brockton and Riabowol for their guidance and constructive criticisms that stimulated my

critical thinking. I also thank my external examiner, Dr. Tina Cheng, for her critical input during

my thesis defence, and Dr. Roman Krawetz for serving as the neutral chair for my M.Sc. defence.

I thank my peers in Dr. Lee’s laboratory (Uilst, Sung-Myung, Alex, Guohong, Vincent, Saranya,

Kwon-Jung) for their friendship and for providing a very supporting environment. I thank the

Saudi Arabia’s King Abdullah Scholarship Program and the Ministry of Education for giving me

the opportunity to continue my education in Canada. Finally, I would like to thank my family for

all their support.

III

Dedication

I dedicate this work to my mother, father, siblings and friends who supported, listened, and encouraged me to continue my studies and complete my M.Sc. degree.

Thank you all for everything

Table of Contents

Abstract ...... II

Acknowledgements ...... III

Dedication ...... IV

Table of Contents ...... V

List of Tables ...... IX

List of Figures and Illustrations ...... X

List of Symbols, Abbreviations and Nomenclature ...... XI

Chapter One: Introduction, Hypothesis and Objective ...... 1

1.1 Introduction ...... 2

1.1.1 Breast cancer ...... 2

1.1.1.1 Breast cancer treatment ...... 3

1.1.2 Tumor necrosis factor superfamily (TNFSF) ...... 4

1.1.2.1 TNF ligands and receptors ...... 4

1.1.2.2 TNFSF function ...... 9

1.1.3 “A proliferation-inducing ligand” (APRIL) is a member of the TNFSF of cytokines ...... 9

1.1.3.1 APRIL gene and protein domains ...... 9

1.1.3.2 APRIL receptors ...... 12

1.1.3.3 APRIL function and signaling ...... 13

1.1.4 PI3K/AKT signaling in cell survival, proliferation and apoptosis...... 14

1.1.4.1 PI3K/AKT signaling ...... 14

1.1.4.2 PI3K/AKT-associated function ...... 16

1.1.5 Signal transducers and activator of transcription 3 (STAT3) signaling ...... 17

1.1.6 Colony stimulating factor 2 receptor beta (CSF2RB) is involved in the stimulation of multiple

haematopoietic cell functions ...... 18

1.2 Hypothesis ...... 20

1.3 Objective ...... 20

Chapter Two: Materials and Methods ...... 21

2.1 Reagents ...... 22

2.2 Cells and cell culture ...... 22

2.3 Plasmids ...... 23

2.4 Transfection of APRIL and/or CSF2RB into T47D or HEK 293 cells ...... 23

2.5 SDS-PAGE and western blotting ...... 24

2.6 Proliferation assay ...... 24

2.7 HEK 293-expressed FLAG-sAPRIL purification: ...... 25

2.7.1 Immunoprecipitation ...... 25

2.7.2 Heparin affinity chromatography ...... 25

2.7.3 DEAE column chromatography ...... 26

2.7.4 Gel filtration chromatography ...... 26

2.7.5 Ammonium sulfate precipitation ...... 26

2.8 GST-sAPRIL expression and purification ...... 27

2.9 Identification of APRIL binding proteins in T47D cells ...... 27

2.9.1 Affinity binding to FLAG-sAPRIL immobilized with anti-FLAG agarose immunoprecipitation reagent 27

2.9.2 Far western blotting ...... 28

2.9.3 Affinity binding to GST-sAPRIL immobilized with GSH-agarose ...... 30

2.10 Immunoprecipitation and coimmunoprecipitation ...... 32

2.11 Test to characterize CSF2RB interaction with APRIL ...... 32

2.12 Analysis of APRIL-mediated signaling in T47D cells ...... 32

2.13 Statistical analysis ...... 33

Chapter Three: RESULTS ...... 34

3.1 To test whether april promotes proliferation of breast cancer cells ...... 35

3.1.1 Transfection of APRIL into T47D breast cancer cells causes increased proliferation...... 35

3.1.2 Purified sAPRIL induces proliferation of T47D breast cancer cells...... 37

3.1.2.1 Expression of sAPRIL in HEK 293 cells...... 37

3.1.2.2 Purification of FLAG sAPRIL from HEK293 cells by FLAG immunoprecipitation ...... 39

3.1.2.3 Effect of the purified sAPRIL on T47D breast cancer cell proliferation ...... 41

3.1.3 An APRIL inhibitory peptide reverses sAPRIL-induced T47D cell proliferation ...... 44

3.2 Purification of sAPRIL ...... 46

3.2.1 Purification of FLAG-tagged sAPRIL from HEK 293 cells by column chromatography...... 46

3.2.1.1 DEAE column chromatography ...... 46

3.2.1.2 Heparin affinity chromatography ...... 47

3.2.1.3 Gel filtration chromatography ...... 51

3.2.1.4 Purification of FLAG-tagged sAPRIL from HEK 293 cells by ammonium sulfate precipitation ..... 53

3.2.2 Purification of GST-tagged sAPRIL by glutathione affinity column chromatography ...... 55

3.3 To identify and characterize the targets of APRIL in breast cancer cells ...... 57

3.3.1 To identify the targets of APRIL in breast cancer cells...... 57

3.3.1.1 Identification of APRIL-interacting proteins by affinity binding to FLAG-sAPRIL immobilized with

anti-FLAG agarose ...... 57

3.3.1.2 Identification of APRIL interacting proteins by far western blotting ...... 61

3.3.1.3 Identification of APRIL interacting proteins by affinity binding to GST-sAPRIL immobilized with

glutathione agarose ...... 67

VII

3.3.2 Classification of identified APRIL interacting proteins in T47D cells ...... 77

3.3.3 Characterization of APRIL interaction with CSF2RB ...... 83

3.3.3.1 Analysis of sAPRIL and CSF2RB interaction by coimmunoprecipitation ...... 83

3.3.3.2 Analysis of sAPRIL and CSF2RB interaction by far western blotting ...... 87

3.4 To determine whether pi3k/akt and stat3 are involved in the proposed april-mediated breast

cancer cell proliferation...... 89

Chapter Four: Discussion and Future Directions ...... 91

4.1 Discussion ...... 92

4.1.1 Impact of APRIL on breast cancer cell proliferation ...... 92

4.1.2 Isolation of sAPRIL ...... 93

4.1.3 Identification of APRIL-interacting proteins in breast cancer cells ...... 96

4.1.4 CSF2RB ...... 97

4.1.5 PI3K/AKT and STAT3 pathways...... 98

4.2 Summary and Future Direction ...... 99

Chapter Five: References ...... 101

VIII

List of Tables

Table 1. Members of the TNF/TNFR superfamily ...... 7

Table 2. APRIL-interacting proteins detected by mass spectrometry of T47D proteins that bind to FLAG-sAPRIL immobilized with anti-FLAG-agarose...... 60

Table 3. APRIL-interacting proteins detected by mass spectrometry of T47D proteins that bind to FLAG-sAPRIL by far western blotting ...... 63

Table 4. APRIL-interacting proteins detected by mass spectrometry of T47D proteins that bind to GST-sAPRIL immobilized with glutathione agarose...... 69

Table 5. Classification of APRIL-binding proteins based on cell function...... 78

List of Figures and Illustrations

Figure 1. Humans APRIL (A) amino acid sequence and (B) protein domains...... 11

Figure 2. Schematic for far western blotting. (Wu and Chen, 2007)...... 29

Figure 3. Flowchart for affinity binding to GST-sAPRIL immobilized with GSH-agarose...... 31

Figure 4. APRIL enhances proliferation of breast cancer cells ...... 36

Figure 5. Generation of HEK 293 cells stably expressing FLAG-tagged soluble APRIL (sAPRIL) ...... 38

Figure 6. Purification of FLAG-tagged sAPRIL by FLAG immunoprecipitation...... 40

Figure 7. Purified sAPRIL promotes proliferation of T47D breast cancer cells ...... 42

Figure 8. An APRIL-specific blocking peptide inhibits APRIL-induced proliferation of breast cancer cells ...... 45

Figure 9. Purification of sAPRIL by (A) DEAE anion exchange chromatography and (B) heparin affinity chromatography ...... 50

Figure 10. Purification of sAPRIL by gel filtration chromatography ...... 52

Figure 11. Isolation of sAPRIL by ammonium sulfate precipitation...... 54

Figure 12. Purification of GST-sAPRIL by glutathione affinity column ...... 56

Figure 13. Isolation of APRIL-interacting proteins in breast cancer cells by affinity binding to FLAG-sAPRIL/FLAG agarose ...... 59

Figure 14. Far western blotting to identify sAPRIL binding proteins ...... 62

Figure 15. Amino acid sequence alignment of (A) CSF2 and APRIL, (B) CSF2RB and BCMA and, (C) CSF2RB and TACI ...... 73

Figure 16. Amino acid sequence alignment of the 2.2 kDa putative APRIL-interacting protein and (A) T cell receptor α and β, (B) BCMA, (C) TACI and (D) CSF2RB...... 75

Figure 17. Immunoprecipitation of sAPRIL and CSF2RB expressed in HEK 293 cells ...... 85

Figure18. Coimmunoprecipitation of FLAG-sAPRIL and CSF2RB-Myc ...... 86

Figure 19. APRIL interacts with CSF2RB directly ...... 88

Figure 20. Activation of STAT3 and AKT following sAPRIL treatment of breast cancer cells . 90

List of Symbols, Abbreviations and Nomenclature

Symbol Definition

Akt AK mouse strain Thymoma

APRIL A proliferation inducing ligand

BAFF B-cell activation factor

BRCA1 Breast cancer 1

BRCA2 Breast cancer 2

CD256 CD256 antigen

CCK8 Cell Counting Kit-8

CLL Chronic lymphocytic leukemia

CRD Cysteine-rich domain

CSF2RB Colony stimulating factor 2 receptor beta

CSF2 Colony stimulating factor 2

C-terminal Carboxy-terminal

DD Death domain

DMEM Dulbecco's Modified Eagle Medium

DTT Dithiothreitol

ER Estrogen receptor

ERK Extracellular signal regulated kinase

FADD Fas associated death domain

FBS Fetal bovine serum

FLAG-sAPRIL FLAG-tagged soluble APRIL

EDTA Ethylenediaminetetraacetic acid

HEK 293 Human embryonic kidney cells 293

IL-3 Interleukin 3

IL-5 Interleukin 5

INPP4B Inositol polyphosphate 4-phosphatase type II

G418 Geneticin

GM-CSF Granulocyte-macrophage colony stimulating

factor

GPI Glycosylphosphatidylinositol

GST glutathione S-transferase

JAK Janus kinase

JUN Jun NH2-terminal kinase (JAK)

NF-kB Nuclear factor kappa B

N-terminal Amino-terminal

PVDF Polyvinylidene difluoride p70S6K Ribosomal protein S6 kinase

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel

electrophoresis

XII

1.1 Introduction

1.1.1 Breast cancer

Breast cancer is the second most common cause of cancer-related death in women, surpassed only by lung cancer (American Cancer Society, Sept 25, 2014). In Canada, it was estimated that 25,000 women were diagnosed with breast cancer in 2015 (Canadian Cancer Society’s Steering

Committee on Cancer Statistics, Canadian Cancer Statistics 2015, Toronto, ON; Canadian Cancer

Society, 2015).

Breast cancer usually arises from the ductal or lobular regions of the breast (American Cancer

Society, Sept 25, 2014). It is categorized into six types based on the molecular biology profile of the tumor: (1) luminal A (50%-60%; ER+, PR-\+, HER2-); (2) luminal B (10-20%; ER+, PR-\+,

-\+ - - + - - HER2 ); (3) HER2-enriched (10-15%; ER , PR , HER2 ); (4) basal-like (10-20%; ER , PR ,

HER2-); (5) normal breast-like (5-10%; ER-\+, HER2-); (6) claudin-low (12-14%; ER-, PR-, HER2-

, CD44, SNAI3) (Eroles et al. 2012). Brewster and Helzlsouer (2001), Russo and Russo (2004),

and Shah et al. (2014) have discussed the numerous factors that increase the risk of developing

breast cancer. The two most important factors that place women under high risk in general are

gender and age. Breast cancer occurs in both genders but is much more common in women than

men. This is primarily due to the physiological differences between men and women. For

example, a woman’s breast undergoes numerous changes in her lifetime and the levels of estrogen

and progesterone hormones are much higher in women than in men. Since both hormones induce

the cell growth and differentiation of breast cells, women’s exposure to estrogen and progesterone

raises the chance of disease. In addition, as women age, the risk of developing breast cancer also

increases. Genetics and family history of breast cancer further increase the risk of developing

breast cancer. The presence of disease among relatives of the first degree such as mother or sister increases the chance of getting breast cancer.

Ten percent of breast cancer is usually associated with gene mutations that are inherited from

parents (Brewster and Helzlsouer 2001, Edlich et al. 2005, Martin and Weber 2000, Shah et al.

2014). Inherited breast cancer occurs as early onset and may be found in multiple locations within

the breast (Martin and Weber 2000). Current studies indicate that the BRCA1 and BRCA2 (Breast cancer 1and Breast cancer 2) tumor suppressor genes are commonly mutated in inherited breast cancer (Brewster and Helzlsouer 2001, Edlich et al. 2005, O’Donovan and Livingston 2010, Shah

et al. 2014). BRCA1 and BRCA2 functions have been associated with maintaining the integrity

of the chromosome structure and DNA repair, respectively (Gudmundsdottir and Ashworth 2006).

Women with BRCA1 and BRCA2 mutations have up to an 85% risk of developing breast cancer

(Brewster and Helzlsouer 2001, O’Donovan and Livingston 2010, Shah et al. 2014).

1.1.1.1 Breast cancer treatment

Although breast cancer is a threatening disease, it is highly curable if diagnosed at an early stage.

In late stages, however, breast cancer is likely to form metastasis to the bone and brain and

therefore, diagnosis at an early stage is critical for increased chance of survival (Scully et al. 2012).

Regular breast self and clinical examination help to detect breast cancer at an early stage (Brewster

and Helzlsouer 2001, Shah et al. 2014).

A variety of treatments are available to fight breast cancer, depending on the stage and nature of

the disease. Surgery (lumpectomy or mastectomy) is a common treatment to remove the tumor

and the surrounding tissue or the entire breast (Shah et al. 2014). This is often followed by radiation or chemotherapy (Shah et al. 2014). Increased knowledge on the nature of various types of breast cancer has aided in the development of newer forms of treatment such as hormone therapy, including the targeting of the estrogen receptor [ER] (Shah et al. 2014, Palmieri et al.

2014). Others have begun to focus on the therapeutic potential of cytokines that regulate breast

cancer cell growth. Some members of the tumor necrosis factor superfamily (TNFSF) of cytokines

have been implicated in breast cancer progression and thus, may be exploited in the development

of new therapies for the disease (Pelekanou et al. 2008).

1.1.2 Tumor necrosis factor superfamily (TNFSF)

The existence of a tumor necrosis factor was first reported in the mid 1970 (Carswell et al. 1975).

Since this initial discovery, the number of tumor necrosis factor superfamily members identified

in humans has increased. The TNFSF is now recognized to consist of 19 ligands with 29 receptors

(Bodmer et al. 2002, Dempsey et al. 2003).

1.1.2.1 TNF ligands and receptors

The known TNF ligands (TNFLs) and TNF receptors (TNFRs) are shown in Table 1 (Bodmer et

al. 2002, Locksley et al. 2001). The TNFLs are generally type II transmembrane proteins with a

single transmembrane domain, an amino-terminal (N-terminal) intracellular domain and a

carboxy-terminal (C-terminal) extracellular domain (Bodmer et al. 2002). The C-terminus corresponds to the TNF homology domain (THD) that has 20-30% amino acid homology to other

TNFSF members, and recognizes the cysteine-rich domains of the TNFRs (Bodmer et al. 2002).

The THDs form a noncovalent trimer to allow interaction of a TNFL with their receptors (Locksley

et al. 2001, Magis et al. 2012). The TNFRs are characterized by the presence of a 30-40 amino acid cysteine-rich domain (CRD) consisting of mostly three disulfide bonds, resulting in an elongated molecule (Locksley et al. 2001, Magis et al. 2012). Among the 29 TNFRs, 22 have a signal peptide and are type 1 with a single transmembrane domain and an N terminal extracellular domain; four (TACI, BCMA, BAFFR, XEDAR) are type III with no signal sequence, a single transmembrane domain and an N terminus extracellular domain; DcR1 is attached to the cell membrane and; OPG and DcR3 receptors are existed as soluble forms (Magis et al. 2012).

TNFRs bind to their ligands with high affinity to form a unique complex structure that activate specific cell signaling pathways (Dempsey et al. 2003, Locksley et al. 2001). They are classified

into 3 groups based on their signaling characteristics and cytoplasmic sequences: (a) those that have a death domain (DD) and activate the caspase pathway, inducing apoptosis (e.g., DR4 /Fas;

Dempsey et al. 2003, Kischkel et al. 2000); (b) those that contain tumor necrosis factor receptor

associated factor (TRAF)-interacting motifs (TIMs) at their cytoplasmic domain which cause

activation of different signal pathways such as phosphoinositide 3-kinase (PI3K), nuclear factor kappa B (NF-kB), p38 MAPK and extracellular signal regulated kinase (ERK) and (Dempsey et al. 2003, Zhang 2004) and (c) those that do not have clear signaling motifs or domains but can play complementary role with the other groups for ligand binding (e.g., DcR1/TRAIL-R3)

(Dempsey et al. 2003).

Another TNFR classification is based on the receptor’s CRD structure (Magis et al. 2012), which

is important for ligand-binding specificity. Thus, the TNFRs are further classified as : (a) type S

(small receptors), containing less conserved CRDs (e.g., BCMA and BAFF-R ); (b) type L-1 (large

receptors) that have CRDs in their N-terminal (e.g., CD40 and FAS )and (c) types L-2, L-3 and L-

4 (large receptors), containing CRDs at various positions other than the N terminal (e.g., CD30 and AITR).

Table 1. Members of the TNF/TNFR superfamily. 1 (Bodmer et al. 2002, Locksley et al.

2001).

LIGAND RECEPTORS

1 TNF- α (TNFSF2) TNFR2 TNFR1

2 FASL (TNFSF6) FAS (TNFRSF6) DcR3

3 VEGI (TNFSF15)

4 TRAIL (TNFSF10) DR4 (TNFRSF10A) DR5 (TNFRSF10B)

DcR1(TNFRSF10C) DcR2 (TNFRSF10D)

OPG (TNFRSF11B)

5 EDA-A1 EDAR

6 RANKL (TNFSF11) RANK(TNFRSF11A) OPG (TNFRSF11B)

7 LT α (TNFSF1) HVEM (TNFRSF14) TNFR2 (TNFRSF1B)

TNFR1 (TNFRSF1A)

8 LTβ ( TNFSF3) LTβR (TNFRSF3)

9 TWEKA (TNFSF12) Fn14

10 LIGHT (TNFSF14) DcR3(TNFRSF6B) HVEM

LTβR (TNFRSF3)

11 CD27L (TNFSF7) CD27 (TNFRSF7)

12 CD30L (TNFSF8) CD30 (TNFRSF8)

13 CD40L (TNFSF5) CD40 (TNFRSF5)

14 4-1BBL (TNFSF9) 4-1BB (TNFRSF9)

15 OX40L(TNFSF4) OX40 (TNFRSF4)

16 AITRL(TNFSF18) AITR (TNFRSF18)

17 APRIL (TNFSF13) TACI (TNFRSF13B) BCMA(TNFRSF17)

18 BAFF (TNFSF13B) TACI(TNFRSF13B) BCMA(TNFRSF17)

BAFFR

19 EDA-A2 XEDAR

20 NGFR (TNFRSF16)

21 Troy (TNFRSF19)

22 DR3(TNFRSF12)

23 DR6

1.1.2.2 TNFSF function

Members of the TNFSF have been demonstrated to regulate various physiological and pathological conditions, including inflammation, homeostasis, autoimmunity, development of the immune system, morphogenesis, rheumatoid arthritis, and diabetes (Aggarwal 2003, Dempsey et al. 2003,

Gaur and Aggarwal 2003, Locksley et al. 2001, Magis et al. 2012, Tansey and Szymkowski 2009,

Wiensa and Glenney 2011). However, TNFSF members have been mostly studied for their roles in regulating cell survival, proliferation, differentiation and apoptosis (Aggarwal 2003, Dempsey et al. 2003, Gaur and Aggarwal 2003, Locksley et al. 2001, Ware 2003). For example, Fas and

TNFR1 pro-apoptotic signaling, through interaction with Fas associated death domain (FADD), is now known to cause the formation of the death-inducing signaling complex (DISC; Dempsey et al. 2003). This results in hierarchical activation of caspase, finally activating the executor caspase, caspase-3, and eventually leading to apoptosis (Dempsey et al. 2003). On the other hand, some

TNFSF members have been shown to have a cell survival and proliferation effect (Dempsey et al.

2003, Gaur and Aggarwal 2003). One proliferation pathway occurs through the activation of NF- kB via the recruitment of TRAFs that interact either directly (through the cytoplasmic TRAF- interacting motifs, TIM) or indirectly with TNFR (Dempsey et al. 2003, Gaur and Aggarwal 2003).

1.1.3 “A proliferation-inducing ligand” (APRIL) is a member of the TNFSF of cytokines

1.1.3.1 APRIL gene and protein domains

APRIL, a member of the TNFSF of cytokines, is also known as TNF-related death ligand-1

(TRDL-1), TNF- And APOL-Related Leukocyte Expressed Ligand 2 (TALL-2), and Tumor

Necrosis Factor (Ligand) Superfamily, Member 13 (TNFSF13; Mhawech-Fauceglia et al. 2006,

Wallweber et al. 2004). Lopez-Fraga et al. 2001and Planelles et al. 2008 described APRIL as 250-

amino acid type II transmembrane protein that consists of a cytoplasmic domain of 28 amino acids

at the N terminal region, a hydrophobic transmembrane domain of 21 amino acids, and an

extracellular domain at the C terminal (Figure 1). It is encoded in human by the APRIL gene located on chromosome 17p13 (Ding et al. 2009, Planelles et al. 2008). The gene is comprised of six exons and three alternatively spliced transcript variants of this gene encoding distinct isoforms,

β, γ and δ, have been reported (Ding et al. 2009, Planelles et al. 2008). The biological roles of

isoforms β and γ are unknown (Planelles et al. 2008). However, the δ form of APRIL has been

identified as an endogenous hybrid transcript of TWEAK and APRIL (TWE-PRIL; Kolfschoten

et al. 2003, Pradet-Balade et al. 2002, Planelles et al. 2008). The translated product is a

transmembrane fusion protein containing the intracellular domain of TWEAK (TNFSF 12;

transmembrane domain and stalk region), and the APRIL extracellular domain, which contains the

binding region for its receptors (Kolfschoten et al. 2003, Pradet-Balade et al. 2002). Thus, TWE-

PRIL is expressed at the cell membrane (Kolfschoten et al. 2003, Pradet-Balade et al. 2002).

Although TWE-PRIL has been found to activate T and B lymphocytes, its true functions are still

unclear (Kolfschoten et al. 2003, Planelles et al. 2008).

APRIL is processed in the Golgi by furin into a mature 16 kDa soluble secreted form and an 11

kDa stalk that remains in the cell (Figure 1B; Lopez-Fraga et al. 2001). In addition, this process is

essential for the biological activity of APRIL (Lopez-Fraga et al. 2001)

1 MPASSPFLLA PKGPPGNMGG PVREPALSVA LWLSWGAALG AVACAMALLT 50

51 QQTELQSLRR EVSRLQGTGG PSQNGEGYPW QSLPEQSSDA LEAWENGERS 100

101 RKRRAVLTQK QKKQHSVLHL VPINATSKDD SDVTEVMWQP ALRRGRGLQA 150

151 QGYGVRIQDA GVYLLYSQVL FQDVTFTMGQ VVSREGQGRQ ETLFRCIRSM 200

201 PSHPDRAYNS CYSAGVFHLH QGDILSVIIP RARAKLNLSP HGTFLGFVKL 250

Figure 1. Humans APRIL (A) amino acid sequence and (B) protein domains. The predicted transmembrane region (TM, blue), the furin cleavage site (red), the glycosylation site (purple), the HSPG binding site( orange), and the TNF homology region (green) are indicated (Hahne et al. 1998, Ingold et al. 2005). NCBI Accession BAE 16556, Version BAE 16556.1, gi: 71480046).

1.1.3.2 APRIL receptors

APRIL shares similarities in sequence, function and receptors with B-cell activation factor

(BAFF), another member of the TNFSF (Hymowitz et al. 2005, Mhawech-Fauceglia et al. 2006,

Planelles et al. 2008, Wallweber et al. 2004). BAFF, also known as BLyS, TALL-1, and

TNFSF13B has 30% sequence similarity with APRIL (Hymowitz et al. 2005, Mhawech-Fauceglia

et al. 2006, Planelles et al. 2008, Wallweber et al. 2004). Both APRIL and BAFF bind to the TNF

receptor superfamily (TNFRSF) members, transmembrane activator and cyclophilin ligand

interactor (TACI, also known as TNFRSF13B) and B-cell maturation antigen (BCMA, also known as TNFRSF17) (Hymowitz et al. 2005, Mhawech-Fauceglia et al. 2006, Planelles et al. 2008,

Wallweber et al. 2004). However, BAFF also binds to its unique receptor, BAFF-R (Hymowitz et al. 2005, Mhawech-Fauceglia et al. 2006, Wallweber et al. 2004).

TACI has high affinity to both BAFF and APRIL through its two extracellular cysteine-rich

domains (CRDs) while BCMA, through its single CRD, has higher affinity to APRIL than BAFF

(Hymowitz et al. 2005). TACI and BCMA contain a DXL motif, which includes a 6-residue

sequence, (F/Y/W)-D-X-L-(V/T) -(R/G), for interaction with BAFF and APRIL (Hymowitz et al.

2005). However, four amino acid positions in BCMA, Y13, I22, Q25, and R27, have been predicted

to impart binding preference for APRIL (Patel et al. 2004).

It has been reported that TACI and BCMA expression is limited to immune cells (Mhawech-

Fauceglia et al. 2008, Planelles et al. 2008), but the fact that APRIL affects growth and survival of non-immune cells (Planelles et al. 2008, Mhawech-Fauceglia et al. 2008, Wallweber et al. 2004)

suggests the presence of an alternative APRIL receptor in these cells. There have been studies

demonstrating that heparan sulfate proteoglycans (HSPGs) in non-immune cells such as fibroblasts

and epithelial cells bind APRIL with high affinity (Hendriks et al. 2005, Ingold et al. 2005). It

was found that the amino acid sequence Q109KQKKQ114 at the N- terminal of mature (secreted)

APRIL, and which is not part of the TNF fold, is required for binding to HSPG but not to TACI

or BCMA (Hendriks et al. 2005, Ingold et al. 2005). Therefore, HSPG does not compete with

TACI or BCMA for binding in APRIL (Hendriks et al. 2005, Ingold et al. 2005). It is still unknown whether HSPG is a true receptor for APRIL but it was hypothesized that HSPGs may serve to facilitate APRIL interaction with a receptor to promote APRIL function (Hendriks et al. 2005).

Thus, a specific receptor for APRIL in non-immune cells remains to be identified.

1.1.3.3 APRIL function and signaling

Although recognized to be expressed in lymphoid cells, APRIL has also been detected in a number of other cell types, including in normal epithelial cells, osteoclasts, tumor-infiltrating neutrophils, melanoma, glioblastoma cell lines, hepatocellular carcinoma and colorectal cells (Deshayes et al.

2004, Planelles et al. 2008, Mhawech-Fauceglia et al. 2006, Okano et al. 2005, Wang G. et al.

2013b). The normal biological function of APRIL is still being examined but in vitro studies in immune cells revealed that APRIL acts together with BAFF as stimulators for T cells (Planelles et al. 2008, Stein et al. 2002, Yu et al. 2000). APRIL was also shown to induce proliferation and survival of Raji B cells through the activation of NF-kB and Jun NH2-terminal kinase (JUK), and

Jurkat T cells by and NF-AT (Yu et al. 2000). However, APRIL was also demonstrated to stimulate apoptosis in Jurkat T cells (Kelly et al. 2000). In B cells, APRIL was further shown to assist in IgA class switch recombination to specific antibody subtypes (Castigli et al. 2004,

Planelles et al. 2008).

In certain types of non-immune cells, APRIL was shown to stimulate proliferation and survival such as in malignant glioblastoma cells, myeloma and colorectal cancer cells (Deshayes et al. 2004,

Ding et al. 2009, Mhawech-Fauceglia et al. 2008, Wang G. et al. 2013b). In vivo, APRIL- transfected tumor cells were found to grow faster in nude mice while knocking down APRIL significantly inhibited tumor cell growth (Wang G. et al. 2013b). APRIL was also found to induce invasion and metastasis of tumor cells, including colorectal cancer cells (Deshayes et al. 2004,

Ding et al. 2009, Wang G. et al. 2013b). In chronic lymphocytic leukemia (CLL) patients, significantly increased level of serum APRIL was detected, suggesting the prognostic value of

APRIL in the survival of CLL patients (Planelles et al. 2008). In addition, treatment with BCMA-

Fc, which binds both APRIL and BAFF, enhances apoptosis of CLL B cells, indicating a role for both cytokines in the survival of these cells (Planelles et al. 2008). Apparently, induction of apoptosis coincides with a reduced activation of NF-κB (Planelles et al. 2008). APRIL was further reported to promote survival of multiple myeloma cells by stimulating G1/S progression (Quinn et al. 2011). Consistent with this observation, depletion of APRIL by siRNA in SW480 cells enhances the cell arrest at G0/G1 phase and apoptosis via obstruction of the TGF-β1/ERK pathway

(Wang et al. 2013a).

1.1.4 PI3K/AKT signaling in cell survival, proliferation and apoptosis.

1.1.4.1 PI3K/AKT signaling

The PI3K/AKT (phosphatidylinositol 3-kinase/AK mouse strain Thymoma) pathway is a critical signaling pathway in all cell types (Cheng et al. 2005, Fresno Vara et al. 2004, Manning and

Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006). It controls many aspects of

cellular functions, including proliferation, survival, apoptosis as well as differentiation, protein

synthesis, metabolism and motility (Cheng et al. 2005, Fresno Vara et al. 2004, Manning and

Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006). Several reports have demonstrated

the influence of the PI3K/AKT pathway in tumor development, making this pathway a potential target for tumor therapy (Cheng et al. 2005, Fresno Vara et al. 2004, Manning and Cantley 2007,

Osaki et al. 2004, Yoeli-Lerner and Toker 2006).

Binding of specific growth factors to their respective cell receptors leads to the activation of PI3K

(Fresno Vara et al. 2004, Osaki et al. 2004). Activated PI3K causes phosphorylation of

phosphatidylinositol 4, 5 biphosphate (PI(4,5) P2) at the 3 position of the inositol ring to generate

phosphatidylinositol 3,4,5-triphosphate PI(3,4,5)P3 (Fresno Vara et al. 2004, Osaki et al. 2004).

The enzyme SH2 domain-containing inositol 5-phosphatase 1/2 (SHIP1/2) can then dephosphorylate PIP3 in the 5 position to produce PI (3,4)P2, which can be dephosphorylated by

inositol polyphosphate 4-phosphatase type II (INPP4B) at position 4 to generate PI(3)P (Agoulnik et al. 2011, Zhang and Claret 2012) . Both PI(3,4,)P2 and PI(3,4,5)P3 are essential second

mssengers for localization and full activation of Akt (Cheng et al. 2005, Fresno Vara et al. 2004,

Manning and Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006, Zhang and Claret

2012).

Akt is also known as protein kinase B (PKB) or related to protein kinase AC (RAC;Cheng et al.

2005, Manning and Cantley 2007, Yoeli-Lerner and Toker 2006). Three isoforms of Akt have

been identified in mammals: Akt1/PKBα, Akt2/PKBβ, and Akt3/PKBγ (Cheng et al. 2005, Fresno

Vara et al. 2004, Manning and Cantley 2007, Yoeli-Lerner and Toker 2006). The isoforms consist

of a pleckstrin homology (PH) domain at the N-terminal, a central kinase domain, and a C-terminal

regulatory domain (Fresno Vara et al. 2004, Osaki et al. 2004).

Full activation of Akt occurs through phosphorylation of residues Thr308 and Ser473 (Ma et al.

2008). Ma et al (2008) reported that PI (3, 4, 5) P3 level correlates with Akt phosphorylation at

Thr308 while PI (3, 4) P2 level correlates with Akt phosphorylation at Ser473. They also

demonstrated that the level of PI (3, 4) P2 determines Akt activity. On the other hand, Akt

phosphorylation is tightly controlled by the two tumor suppressor proteins, PTEN (phosphatase

and tensin homolog) and INPP4B (Cheng et al. 2005, Fedele et al. 2010, Fresno Vara et al. 2004,

Gewinner et al. 2009, Manning and Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006,

Zhang and Claret 2012). PTEN exerts enzymatic activity as a PI (3, 4, 5) P3 phosphatase, and as

indicated above, INPP4B serves as a PI (3, 4) P2 phosphatase, and thus, both phosphatases oppose

the activity of PI3K (Cheng et al. 2005, Fedele et al. 2010, Fresno Vara et al. 2004, Gewinner et

al. 2009, Manning and Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006, Zhang and

Claret 2012). Together, the above demonstrates the interplay between the modulators of the PI3K-

Akt pathway.

1.1.4.2 PI3K/AKT-associated function

Akt modulates the function of its substrates involved in regulating cell cycle progression, proliferation, survival and apoptosis (Cheng et al. 2005, Fedele et al. 2010, Fresno Vara et al. 2004,

Gewinner et al. 2009, Manning and Cantley 2007, Osaki et al. 2004, Yoeli-Lerner and Toker

2006). Akt regulation of apoptosis is achieved through direct or indirect activation of its downstream targets such as Bad (Cheng et al. 2005, Fresno Vara et al. 2004, Manning and Cantley

2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006). For cell growth and proliferation, phosphorylation of Akt by phosphoinositide-dependent kinase-1 (PDK1) activates the mammalian target of rapamycin (mTOR), leading to phosphorylation of ribosomal protein S6 kinase (p70S6K), a regulator of translationand (Cheng et al. 2005, Fresno Vara et al. 2004, Manning and Cantley

2007, Osaki et al. 2004, Yoeli-Lerner and Toker 2006).

Many studies have demonstrated that alterations in the PI3K/Akt pathway are associated with cancer, including breast cancer (Osaki et al. 2004). For example, loss of function of PTEN or

INPP4B, a negative regulators of Akt, were observed in breast cancer cases (Osaki et al. 2004).

1.1.5 Signal transducers and activator of transcription 3 (STAT3) signaling

STAT3, a transcription factor, is a member of the signal transducer and activator of transcription

(STAT) protein family that consists of 7 members: STAT1, STAT2, STAT3, STAT4, STAT5A,

STAT5B and STAT6 (Levy and Lee 2002, Siveen et al. 2014). STAT3 is involved in controlling

several cell functions, including cell proliferation, survival, differentiation, metabolism and

immunity (Levy and Lee 2002, Siveen et al. 2014). STAT3 can be activated by cytokines and

growth factors where it becomes phosphorylated by JAK (Janus kinase) at the tyrosine 705 residue

(Levy and Lee 2002, Siveen et al. 2014). In breast cancer, STAT3 induces expression of genes

that are involved in cell proliferation such as cyclin D1, and CDC2 (Banerjee and Resat 2016,

Siveen et al. 2014).

1.1.6 Colony stimulating factor 2 receptor beta (CSF2RB) is involved in the stimulation of

multiple haematopoietic cell functions

The PI3K/AKT as well as the STAT3 signaling pathways have been associated with CSF2RB

signaling (Broughton et al. 2012, de Groot et al. 1998, Guthridge et al. 1998, Hercus et al. 2013).

CSF2RB is the common beta chain of the receptor for the cytokines: colony stimulating factor 2

(CSF2 or granulocyte-macrophage colony stimulating factor, GM-CSF), interleukin 3 (IL-3) and

interleukin 5 (IL-5) (Broughton et al. 2012, Guthridge et al. 1998, Hercus et al. 2013). CSF2RB

is a type I cytokine membrane receptor (Murphy and Young 2006). CSF2RB and its ligands have

been implicated in a number of biological functions, innate immune response (Broughton et al.

2012) and certain signal transduction pathways [e.g., ras (Broughton et al. 2012), and epidermal

growth factor receptor (Burge et al. 2001)]. CSF2RB has further been linked to the pathogenesis

of leukemia and allergic and inflammatory diseases such as rheumatoid arthritis and asthma

(Broughton et al. 2012, Murphy and Young 2006), and pulmonary alveolar proteinosis (PAP;

Broughton et al. 2012, Dirksen et al. 1997).

CSF2RB forms part of a cytokine receptor heterodimer that consists of two subunits: an alpha chain (CSF2Rα, IL3Rα or IL5Rα) that is exclusive to the ligand (CSF2, IL3 and IL5, respectively) and the CSF2RB beta chain (βc) that is common to the CSF2, IL3 and IL5 receptors

(Broughton et al. 2012, de Groot et al. 1998, Guthridge et al. 1998, Hercus et al. 2013). CSF2RB functions together with the two ligand-specific α-subunits complex (Hercus et al. 2013).

Structural analysis by Broughton et al. (2015) revealed that CSF2RB consists of four fibronectin type III domains that intertwine to form a homodimer.

Ligand interaction with CSF2RB leads to receptor oligomerization that facilitates binding of Janus kinases (JAK) and transphosphorylation. Activation of JAK causes phosphorylation of CSF2RB at tyrosine 577, 612, 695 and 750 (Broughton et al. 2015, de Groot et al. 1998, Hercus et al. 2013) which initiates multiple downstream pathways such as PI3K and STAT3, inducing biological effects such as cell survival and proliferation (Broughton et al. 2012, Guthridge et al. 1998, de

Groot et al. 1998, Hercus et al. 2013).

1.2 Hypothesis

My hypothesis is that APRIL interacts with a novel breast cancer cell target, activates the

PI3K/AKT and STAT3 pathways, and promotes breast cancer cell proliferation.

1.3 Objective

My objective is to identify the molecular mechanism by which APRIL promotes proliferation of breast cancer cells. My specific aims are: 1. To test whether APRIL promotes proliferation of breast cancer cells.

2. To identify and characterize APRIL interacting proteins in breast cancer cells.

3. To determine whether PI3K/AKT and STAT3 are involved in the proposed APRIL-

mediated breast cancer cell proliferation.

2.1 Reagents

Recombinant FLAG-tagged soluble APRIL (FLAG-sAPRIL; AG-40B-0017-3010) was obtained from Adipogen. The chromatography materials used and their respective sources were: HiTrap

DEAE FF, HiTrap Heparin HP and Superdex 200 Increase 10/300 GL columns were from GE

Healthcare Life Sciences. The APRIL blocking peptide (AAAPLAQPHMWA; He et al. 2015) was synthesized at the University of Calgary’s DNA services core facility. The FLAG immunoprecipitation kit, GSH-agarose and ammonium sulfate were from Sigma. Aquacide III and centricon were from Millipore. SYPRO Ruby stain was from Molecular Probes. The

antibodies used and their respective sources were: APRIL (Aprily-5), Novus Biologicals; APRIL

(ED), Axxora; CSF2RB, eBioscience; FLAG M2, Sigma; Myc, Santa Cruz Biotech.; STAT3

pTyr705 and STAT3, AKT pT308, AKT pS473 and AKT, Cell Signaling. The cell counting kit-

8 (CCK-8) was from Dojindo Molecular Tech. Inc. Coomassie brilliant blue was from Bio-Rad.

2.2 Cells and cell culture

Breast cancer cells (T47D) and human embryonic kidney cells (HEK 293) were obtained from the

American Type Culture Collection (ATCC; Rockville, MD). These cell lines are also described

by ATCC. Breast cancer cells and HEK293 were cultured and maintained in Dulbecco's Modified

Eagle Medium (DMEM; Thermo-Fisher Scientific). Culture media were supplemented with 10%

heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific), 100U penicillin and 0.1

mg/ml streptomycin (GIBCO) at 37°C in 5% CO2. Neutrophils were isolated from the peripheral

blood of normal and healthy volunteers that gave informed consent.

2.3 Plasmids

The sAPRIL (PS429) and full-length APRIL (PS938) plasmids were kindly provided by Dr. Pascal

Schneider, University of Lausanne, Switzerland. Generation of these plasmids has been described

previously by Hahne et al. (1998). Briefly, the full-length APRIL gene (EMBL/GenBank/DDBJ

accession no. AF046888) was contained in the expressed sequence tag (EST) clone AA292304

that was used to amplify the APRIL coding region using a 5′ forward primer (5′-

CCAGCCTCATCTCCTTTCTTGC-3′) flanked by an EcoRI site and a 3′ reverse primer (5′-

TCACAGTTTCACAAACCCCAGG-3′) flanked by an XbaI site. The amplified fragment was then cut using EcoRI/XbaI and cloned into the mammalian expression vector pCR3 (Invitrogen) in frame with a FLAG peptide at the amino terminal. Soluble APRIL (sAPRIL) was generated using a forward primer (5′-AAACAGAAGAAGCAGCACTCTG-3′) flanked by a PstI site and a

3′ reverse primer (5′-TCACAGTTTCACAAACCCCAGG-3′) flanked by an XbaI site. Thus amplified fragment was cut using PstI/XbaI and cloned into pCR3 in frame with a FLAG peptide at the amino terminal and containing a hemagglutinin signal for protein secretion in eukaryotic cells. The Myc-tagged human CSF2RB ORF mammalian expression plasmid (pCMV3-CSF2RB-

Myc) was obtained from Sino Biological Inc.

2.4 Transfection of APRIL and/or CSF2RB into T47D or HEK 293 cells

T47D cells were stably transfected with pCR3-FLAG-sAPRIL, pCR3-FLAG full-length APRIL or empty pCR3 vector using Lipofectamine 2000 (Invitrogen). Since the plasmids contain a neo gene that encodes neomycin transferase, geneticin (G418; Invitrogen) was used (at a concentration of 250 ug/ml, the optimal concentration determined from titration/killing curve) to select for transfected cells, which are resistant to the antibiotic. The antibiotic-resistant clones were

maintained in media containing 50 ug/ml of G418. HEK 293 cells were also stably transfected with pCR3- FLAG-sAPRIL, pCR3-FLAGfull-length APRIL or empty pCR3 vector using lipofectamine 2000 but transfected cells were selected using 600 ug/ml G418 (the optimal concentration determined from titration/killing curve). Media containing 200 ug/ml of G418 was used to maintain the antibiotic-resistant clones. HEK 293 cells that are either non-transfected or stably transfected with FLAG-sAPRIL were also transiently transfected with CSF2RB-Myc and/or

FLAG-sAPRIL using lipofectamine 2000. HEK 293 cells stably transfected with FLAG-sAPRIL and transiently co-transfected with CSF2RB-Myc were maintained in the presence of 50 ug/ml hygromycin. Transfection efficiency was assessed by western blotting.

2.5 SDS-PAGE and western blotting

Cell culture media, cell lysates or immunoprecipitates were prepared using our lab’s established methods. Protein samples were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes that were blocked with 5% skim milk in PBST (pH 7.4, containing 115 mM NaCl, 16 mM Na2HPO4, 4 mM KH2PO4

and 0.05%Tween-20) for one hour followed by washing and incubation with the primary antibody

for the protein of interest for either 1-2 hours at room temperature or overnight at 4°C. Membranes were then washed and incubated with a secondary antibody for 40 minutes at room temperature.

Immunoreactive bands were detected using the ECL reagent (Amersham).

2.6 Proliferation assay

Cells were seeded at a density of 5 x 103 or 1 x 104 cells/well in a 96-well plate in DMEM

containing 10% FBS, 100U penicillin and 0.1 mg/ml streptomycin. The next day, the culture media

was replaced with low serum (2% FBS) DMEM with or without recombinant FLAG-sAPRIL (5,

10, 20, 40 or 80 ng/ml) that I purified or obtained from Adipogen. Cell viability was assessed for

up to 7 days using CCK-8. To validate the APRIL-induced effect on proliferation of T47D cells,

the effect of APRIL (730 nM) that was blocked by an APRIL-specific blocking peptide,

AAAPLAQPHMWA (20 and 40 nM), was examined. Cell viability was measured using CCK-8.

2.7 HEK 293-expressed FLAG-sAPRIL purification:

2.7.1 Immunoprecipitation

The culture media of FLAG-sAPRIL-transfected HEK 293 cells containing secreted FLAG-

sAPRIL was collected, treated with protease inhibitor cocktail, centrifuged at 14,0000 rpm for 1

hr and filtered using a millipore 0.45 um low protein binding filter. The filtrate containing FLAG-

sAPRIL was incubated with anti-FLAG agarose overnight at 4°C. The FLAG- sAPRIL-/anti-

FLAG agarose complex was isolated by centrifugation at 5000 x g for 30 seconds and washing 3

times with wash buffer (0.5M Tris HCL, pH 7.4, with 1.5 M NaCl). FLAG-sAPRIL was eluted from the complex using 3X FLAG peptide. Purity was assessed by SDS-PAGE and Coomassie brilliant blue staining, a confirmation of protein isolation was determined by western blotting.

2.7.2 Heparin affinity chromatography

Culture media containing FLAG-sAPRIL secreted by transfected HEK 293 cells was collected,

centrifuged and filtered as described in section 2.8a. The filtrate was concentrated using aquacide

III, dialyzed in binding buffer (10 mM sodium phosphate, pH 7) and loaded into a HiTrap™

heparin HP column. Heparin binding proteins were eluted using a gradient of NaCl, in 10 mM

sodium phosphate, pH 7.0. Fraction samples were collected and analyzed by SDS-PAGE and

staining with SYPRO Ruby stain, and western blotting.

2.7.3 DEAE column chromatography

Culture media containing FLAG-sAPRIL secreted by transfected HEK 293 cells was prepared as

described in section 2.8a. The concentrated sample was dialyzed in 20 mM Tris-HCl, pH 9, and

loaded into a HiTrap DEAE FF column attached to an FPLC system. DEAE binding proteins were

eluted using a concentration gradient of NaCl. Fraction samples were analyzed by SDS-PAGE and staining with SYPRO Ruby stain, and western blotting.

2.7.4 Gel filtration chromatography

Culture media containing FLAG-sAPRIL secreted by transfected HEK 293 cells was prepared as described in section 2.8a. The sample was concentrated using centricon-10 and dialyzed in 0.01

M phosphate buffer, pH 7.4, containing 0.14 M NaCl. The sample was then loaded into a Superdex

200 Increase 10/300 GL column attached to an FPLC system. Fraction samples were run on SDS-

PAGE and stained with SYPRO Ruby stain and analyzed by western blotting.

2.7.5 Ammonium sulfate precipitation

Culture media containing FLAG-sAPRIL secreted by transfected HEK 293 cells was prepared as described in section 2.8a. Increasing amount of solid ammonium sulfate was added into the media, stirring the mixture for 20 minutes at 4°C and subjecting to centrifugation at 10000 rpm for 20 min. before any further addition of ammonium sulfate. Precipitates were collected and samples were diluted in distal water were analyzed by SDS-PAGE gel and staining with SYPRO Ruby stain, and western blotting.

2.8 GST-sAPRIL expression and purification

PS429 was used to generate sAPRIL cDNA. The PCR reaction was carried out using the forward

primer, sAPRIL-BamHI-F: GATGATGGATCCAAACAGAAGAAGCAGCACTCTG, and

reverse primer, sAPRIL-NotI-F: GATGATGCGGCCGCTCACAGTTTCACAAACCCCAGG.

The PCR product was cloned into p-GEX-6P-1 (glutathione S-transferase (GST) vector) and

transformed into E.coli (BL21). Expressed GST-sAPRIL was purified from bacterial lysate using

glutathione-agarose in 10 mM phosphate buffer, pH 7.4, containing 150 mM NaCl, and eluted using reduced glutathione.

2.9 Identification of APRIL binding proteins in T47D cells

2.9.1 Affinity binding to FLAG-sAPRIL immobilized with anti-FLAG agarose immunoprecipitation reagent

Initially, culture media of FLAG-sAPRIL transfected HEK 293 cells containing secreted FLAG-

sAPRIL was prepared as described in section 2.8a. The filtrate was then incubated with anti-

FLAG agarose to allow binding of FLAG- sAPRIL to T47D cell lysate, which was pre-cleared by

incubation with anti-FLAG agarose for 20 min at 4°C, was then added to the FLAG-sAPRIL/anti-

FLAG agarose complex and incubated overnight at 4°C. The “APRIL binding protein/FLAG- sAPRIL/anti-FLAG agarose complex” was isolated by centrifugation at 5000 x g for 30 seconds and washing 3 times with wash buffer. The sAPRIL interacting proteins were eluted via a step- gradient concentration of NaCl (200, 300, 400 and 500 mM). Eluted proteins were visualized by

Coomassie brilliant blue, cut off from the gel and identified by mass spectrometry.

2.9.2 Far western blotting

A flow chart for the far western blotting procedure is shown in Figure 2. This method was also

used to identify APRIL binding protein(s) in T47D cells. Lysates (100ug) of T47D cells or

CSF2RB-transfected HEK 293 cells were resolved by SDS-PAGE and transferred to a

polyvinylidene difluoride (PVDF) membrane. The transferred proteins were used as prey. Proteins

on the membrane were denatured and renatured using decreasing concentrations of guanidine-

HCL: 6M, 30 minutes, RT; 3M, 30 minutes, RT; 1M 30 minutes, RT; 0.1M, 30 minutes 4oC and

1h at 4oC with no guanidine-HCL. The membrane was then blocked with 5% skim milk in PBST

then incubated overnight with the bait protein, recombinant FLAG-sAPRIL (5ug/ml), in protein binding buffer, which contains 20 mM Tris (pH 7.6), 0.5 mM ethylenediaminetetraacetic acid

(EDTA), 100 mM NaCl, 1 mM dithiothreitol (DTT), 0.1% Tween-20, 10% glycerol and 2% skim milk. The following day, the membrane was washed 3 times (10 minutes per wash) then incubated with primary antibody to APRIL (APRILy-5 at 4oC overnight, then washed again and incubated

with secondary antibody for 40 min. Immunoreactive bands were detected using the ECL reagent

(Amersham). Immunoreactive bands detected in T47D lysates (prey) that were exposed to APRIL

(bait), but undetected in lysates that were unexposed to APRIL, were considered as potential

APRIL-interacting proteins. These APRIL-interacting candidates were cut off from the membrane, re-run on SDS-PAGE, stained with Coomassie brilliant blue, cut off from the gel and identified by mass spectrometry.

Figure 2. Schematic for far western blotting. (Wu and Chen, 2007).

2.9.3 Affinity binding to GST-sAPRIL immobilized with GSH-agarose

Figure 3 shows a flow chart for affinity binding to GST-sAPRIL immobilized with GSH-agarose.

A lysate of E. coli expressing GST-sAPRIL was incubated with GSH-agarose for 2 hours at 4°C.

After washing with 10 mM phosphate buffer (pH 7.4, containing 150 mM NaCl), T47D cell lysate was added and incubated overnight at 4°C. Unbound proteins were washed off with the phosphate buffer while bound proteins were eluted by incubation with reduced glutathione for 30 minutes at

4°C. Eluted samples were resolved by SDS-PAGE. The gel was stained with Coomassie brilliant blue and candidate APRIL binding proteins (which were not observed in the control sample: GSH- agarose incubated with a lysate of E. coli containing an empty GST vector) were cut and sent for mass spectrometry for identification.

Figure 3. Flowchart for affinity binding to GST-sAPRIL immobilized with GSH-agarose.

2.10 Immunoprecipitation and coimmunoprecipitation

Lysates of transfected (HEK 293/FLAG-sAPRIL; HEK 293/CSF2RB-Myc; and HEK 293/FLAG- sAPRIL/CSF2RB-Myc) and non-transfected HEK293 cells were prepared. Immunoprecipitation of FLAG-sAPRIL and CSF2RB-Myc from cell lysates was done using anti- FLAG M2 and anti-

Myc antibody, respectively. Effectiveness of immunoprecipitation was assessed by western blotting with anti-APRIL (Aprily-5) and anti-CSF2RB, respectively. Coimmunoprecipitation was tested of using sAPRIL or Myc antibody and immunoblotting with FLAG or CSF2RB antibody.

2.11 Test to characterize CSF2RB interaction with APRIL

Interaction between APRIL and CSF2RB was assessed by coimmunoprecipitation as described

above. To determine whether CSF2RB interacts directly with APRIL, lysates of HEK293 cells

transfected with CSF2RB were resolved by SDS-PAGE and transferred to a nitrocellulose

membrane. The membrane with the transferred proteins (prey) was then subjected to far western

blotting using FLAG-sAPRIL as prey (Adipogen).

2.12 Analysis of APRIL-mediated signaling in T47D cells

T47D cells were seeded in 35 mm dishes in DMEM containing 10% FBS, and 100U penicillin and

0.1 mg/ml streptomycin. The following day, cells were treated with 50 ng/ml FLAG-sAPRIL in

low serum (2%FBS) media then harvested at different time points (0, 1, 5, 10, 15 and 30 min and,

1, 24, 48 and 72 hrs) after treatment with APRIL. Protein determination of cell lysates was performed using the BioRad assay kit and 30 ug samples were resolved by SDS-PAGE and western blotting for total and phospho AKT and STAT3 as indicated.

2.13 Statistical analysis

All experiments were repeated at least three times and results were analyzed using student’s t-test. Statistical significance was set at .

p ≤ 0.05

3.1 To test whether april promotes proliferation of breast cancer cells

3.1.1 Transfection of APRIL into T47D breast cancer cells causes increased proliferation.

Preliminary studies in our lab showed that treatment of T47D breast cancer cells with a commercial

recombinant sAPRIL (Adipogen) induced proliferation of these cells. Indeed, previous studies

have demonstrated that exogenous APRIL causes increased proliferation of immune B cells (Yu

et al. 2000, Planelles et al. 2008) and certain types of cancer cells such as colorectal cancer cells

(Wang et al. 2013). However, other studies have also shown that APRIL promotes cell apoptosis

(Roth et al. 2001). To further investigate the effect of APRIL on breast cancer cells, T47D cells

were transfected with full-length APRIL. As serum contains a number of growth promoting

factors, transfected cells were cultured in low serum (2%) media to facilitate detection of a

potential proliferative effect of APRIL in breast cancer cells. The number of viable cells at specific

time points in culture was measured using a colorimetric cell counting method (Cell Counting Kit-

8, CCK8). As shown in Figure 4, T47D cells transfected with APRIL showed a significant increase

(p<0.05) in proliferation on days 5 and 7 in culture compared to control cells transfected with an

empty vector, i.e., after 5 and 7 days in low serum media, the number of T47D cells transfected with APRIL was 1.9 and 2.2 times higher, respectively, than cells transfected with an empty vector.

This result indicates that APRIL promotes proliferation of T47D breast cancer cells.

Figure 4. APRIL enhances proliferation of breast cancer cells. T47D cells were transfected with pCR3-full length APRIL or an empty pCR3 vector as described in Materials and Methods. Cells were grown in media containing 10% FBS overnight then switched to a low serum containing media (2% FBS) for 7 days. Cell number was determined at the indicated time points using CCK8. Values are means ±SD of 3 replicates of a representative experiment, n=3, p<0.05.

3.1.2 Purified sAPRIL induces proliferation of T47D breast cancer cells.

To support my finding above, I sought to further examine the effect of purified sAPRIL on T47D breast cancer cell proliferation. To do so, I initially expressed and purified sAPRIL from a mammalian cell expression system.

3.1.2.1 Expression of sAPRIL in HEK 293 cells.

HEK 293 cells were stably transfected with FLAG-tagged sAPRIL as described in Materials and

Methods. Several stably transfected clones of HEK 293 cells expressing FLAG-tagged sAPRIL were generated. SDS-PAGE and western blotting using an antibody to APRIL showed a 16 kDa immunoreactive band in the culture media of three representative clones of transfected cells

(Figure 5, HEK293-FLAG-sAPRIL, C1 to C3). The 16 kDa immunoreactive band corresponds to sAPRIL detected in neutrophils, which served as positive control for APRIL detection. No APRIL immunoreactive band was detected in media alone or in the culture media of non-transfected HEK

293 cells.

Figure 5. Generation of HEK 293 cells stably expressing FLAG-tagged soluble APRIL (sAPRIL). HEK 293 cells were transfected with pCR3-FLAG-sAPRIL as described in Materials and Methods and grew in 10% FBS. Media from three G418-resistant clones were examined by SDS-PAGE and western blotting for APRIL (APRILY-5). Media alone and media from non- transfected HEK 293 cells were used as negative controls. Neutrophil lysate (10 ug) was used as positive control. “APRIL” is directed at full-length APRIL; * points to an intermediate processed product of APRIL.

3.1.2.2 Purification of FLAG sAPRIL from HEK293 cells by FLAG immunoprecipitation

Isolation of FLAG-tagged sAPRIL from the culture media of FLAG-sAPRIL-expressing HEK 293 cells was performed by FLAG immunoprecipitation as described in Materials and Methods, and according to the manufacturer’s recommendation (Sigma). The isolated protein was resolved by

SDS-PAGE and visualized by Coomassie brilliant blue (Bio-Rad) to assess purity. As shown in

Figure 6A, a 16 kDa band was isolated by FLAG immunoprecipitation and elution using a 3X

FLAG peptide. The 16 kDa band, however, was undetectable in the original sample (input). It was not also detected in the flowthrough from the FLAG immunoprecipitation column as well as in the washes. A considerable amount of a 67 kDa protein, most likely, albumin from the serum added to the HEK 293 culture media, was observed. To confirm the identity of the 16 kDa band, western blotting was performed using an APRIL antibody. Figure 4B shows a 16 kDa APRIL immunoreactive band in the 3X FLAG peptide eluate that corresponds to sAPRIL detected in neutrophils, indicating that the isolated protein was sAPRIL.

Figure 6. Purification of FLAG-tagged sAPRIL by FLAG immunoprecipitation. Culture media of HEK 293 cells transfected with FLAG-sAPRIL was subjected to FLAG immunoprecipitation. FLAG-sAPRIL bound to the FLAG antibody was eluted using a 3X FLAG peptide. (A). Coomassie staining of the sample used for FLAG immunoprecipitation, samples obtained during immunoprecipitation, and after 3X peptide elution. (B). Western blotting of the 3X FLAG peptide eluate in “A” using an antibody to APRIL (Aprily-5). Neutrophils (2 ug) was used as control.

3.1.2.3 Effect of the purified sAPRIL on T47D breast cancer cell proliferation

Since I found that APRIL-transfected T47D breast cancer cells grown in low serum media showed

a clear increase in proliferation (compared to control cells) after 5 and 7 days in culture, I sought

to examine whether my purified sAPRIL can induce T47D breast cancer cell proliferation after 7

days as well. Since preliminary studies in our laboratory showed that 80 ng/ml of commercial

sAPRIL was sufficient to induce breast cancer cell proliferation, I tested the functionality of my

purified sAPRIL using the same concentration. As shown in Figure 7A, after 7 days in low serum

media, the number of T47D cells cultured in the presence of sAPRIL was more than twice number

of cells than that of the T47D cells cultured without sAPRIL, indicating that my purified sAPRIL

promotes cell proliferation and that it is functional.

I then tested whether T47D cells will show a dose-dependent response to my purified sAPRIL.

Figure 7B shows that while serum-deprived T47D cells that were not treated with sAPRIL showed

no measurable increase in cell number after 7 days in culture, serum-deprived T47D cells treated with purified sAPRIL showed a dose-dependent increase in proliferation after 7 days in culture

with maximum response observed at 40 ng/ml. My result shows that the proliferation response to

APRIL treatment starts to plateau at this concentration. Together, these findings indicate that sAPRIL promotes proliferation of T47D breast cancer cells, and concur with: (a) an earlier observation in our laboratory which showed that commercially available recombinant APRIL stimulates breast cancer cell proliferation, and (b) my observation that APRIL-transfected T47D cells show increased proliferation compared to control T47D cells transfected with an empty vector.

Figure 7. Purified sAPRIL promotes proliferation of T47D breast cancer cells. T47D cells were seeded in 96-well plates at a density of 1 X104 cells per well in 10% FBS-containing media as described in Materials and Methods. Following overnight culture, media was changed (day 0) to low serum (2% FBS)-containing media ± sAPRIL at (A) 80 ng/ml or (B) at various concentrations. Media ± sAPRIL was changed every 2 or 3 days. Cell viability in A and B was

assessed using CCK-8 after 7 days of culture in low serum media ± APRIL. Values are means ±SD from 4 replicates of a representative experiment, n=5. Statistical significance was set at p<0.05.

3.1.3 An APRIL inhibitory peptide reverses sAPRIL-induced T47D cell proliferation

Recently, a study by He et al. (2015) demonstrated that a synthesized sAPRIL-BP1 peptide

(AAAPLAQPHMWA) has high binding affinity to sAPRIL and has the ability to inhibit the

proliferative effect of sAPRIL in colorectal cancer cells. Thus, I tested whether sAPRL-BP1 can

also inhibit the proliferative effect of sAPRIL in breast cancer cells. sAPRIL (Adipogen) was pre-

incubated with sAPRIL-BP1 peptide (20 nM or 40 nM) at 37 °C for for 5 minutes. T47D cells in

low serum media were then treated with blocked or unblocked sAPRIL and proliferation was

measured after 3 days. As shown in Figure 8, treatment with sAPRIL caused approximately 1.6

times increase in proliferation of T47D cells. Upon addition of 20 nM sAPRIL-BP1, the

proliferation effect of sAPRIL was reduced but did not reach significance. However, sAPRIL-

BP1 at 40 nM significantly (p < 0.05) blocked the proliferation effect of sAPRIL in T47D cells,

indicating the specificity of APRIL-induced proliferation in these cells. Altogether, my findings

have established that APRIL promotes proliferation of breast cancer cells, at least in the T47D

breast cancer cell model.

Figure 8. An APRIL-specific blocking peptide inhibits APRIL-induced proliferation of breast cancer cells. T47D cells were cultured in the presence or absence of sAPRIL (730nM; Adipogen) and/or the APRIL inhibitory peptide, sAPRIL-BP1 (20 or 40 nM) in serum starved condition (2% FBS) for three days. Values are means ±SD from a representative triplicate experiment, n=5, p< 0.05.

3.2 Purification of sAPRIL

I aimed to use purified sAPRIL as a tool to identify and characterize the targets of APRIL in

breast cancer cells, and to examine the signaling mechanism involved in APRIL-induced

proliferation of breast cancer cells (Chapter III). However, although I have successfully purified

FLAG-sAPRIL from HEK 293 cells by FLAG immunoprecipitation, the yield was remarkably

low. Therefore, I sought to utilize alternate methods to purify sAPRIL.

3.2.1 Purification of FLAG-tagged sAPRIL from HEK 293 cells by column chromatography

Culture media of HEK 293 cells containing secreted FLAG-sAPRIL was collected and prepared

for chromatography as described in Materials and Methods. Samples were then analyzed.

3.2.1.1 DEAE column chromatography

Sample in 20 mM Tris-HCl, pH 9 was loaded into a HiTrap DEAE FF column attached to an FPLC

system. DEAE binding proteins were eluted using a concentration gradient of NaCl as described

in Materials and Methods. As shown in Figure 9A, SYPRO Ruby staining of column fractions

analyzed by SDS-PAGE showed a 16 kDa band, which corresponds to the molecular size of

sAPRIL, in the sample media (lane 1) but there was no obvious presence of a 16 kDa band in the

NaCl eluates. Western blotting using an antibody to APRIL (Figure 9A, bottom panel) confirmed

that sAPRIL was in the sample media but sAPRIL was also detected in the flowthrough, and in

the 50mM, 250mM, 300 mM and 1M NaCl eluates. In Figure 9A (top panel), a strong 67 kD band

[corresponding to the 67 kDa band observed by purifying FLAG-sAPRIL using FLAG

immunoprecipitation (Figure 4), and which is most likely to be albumin from the bovine serum

(BSA) in the media] was detected in the sample media, the flowthrough and the 50mM to 250mM

NaCl eluates. Thus, it appears that the large amount of BSA in the sample competed with sAPRIL for DEAE binding. In addition, sAPRIL that bound to the DEAE column mostly coeluted with

BSA at around 250 mM NaCl.

3.2.1.2 Heparin affinity chromatography

Since BSA coelutes with sAPRIL from a DEAE column and the two proteins cannot be separated

using this type of anion exchange chromatography, I sought to isolate sAPRIL using a heparin

affinity column. Sample in 10 mM sodium phosphate buffer (pH 7.0) was loaded into a HiTrap™

heparin HP column and heparin binding proteins were eluted using a NaCl gradient. Figure 9B, top panel, shows a SYPRO Ruby staining of column fractions analyzed by SDS-PAGE. While there appeared to be a 16 kDa band in the sample media (lane 1) and in the flowthrough (lane 4), a 16 kDa band was not observed in the NaCl eluates. Western blotting using an antibody to APRIL confirmed that sAPRIL was in the flowthrough (Figure 9B, bottom panel) but was not present in the NaCl eluates from the heparin column. The 67 kDa BSA was detected in the sample media, concentrated sample and filtrate, flowthrough from the heparin column, and the 50mM to 150mM

NaCl eluates (lanes 1-13). Therefore, as with the DEAE column, it appears that BSA and other proteins in the sample competed with sAPRIL for heparin binding, and thus, sAPRIL was primarily detected in the flowthrough.

Considering the large amount of BSA in the sample and the limited binding capacities of DEAE and heparin, I used larger volumes of these resins (1 or 5 ml media to 1 or 5 ml resin vs 10 ml media to 50 ml resin). However, the sAPRIL purification pattern, level of BSA contamination and yield did not considerably improve. I also tried connecting the DEAE and heparin columns but

BSA contamination was still significant and the yield was similarly low. I further tried culturing the sAPRIL-expressing HEK 293 cells in low serum (2%) media without phenol red, which also appeared to cofractionate with sAPRIL in the chromatography procedures. However, BSA contamination persisted and sAPRIL yield was still low.

Figure 9. Purification of sAPRIL by (A) DEAE anion exchange chromatography and (B) heparin affinity chromatography. Column eluates were analyzed by SDS-PAGE and SYPRO Ruby staining (A and B, top panels) and western blotting (A and B, bottom panels) using an antibody to APRIL. APRIL-containing neutrophils was used as positive control. Original sample media used: 5 ml for DEAE column and 5 ml for heparin column; column volume, DEAE=1ml; heparin =1ml.

3.2.1.3 Gel filtration chromatography

Since sAPRIL did not bind to the heparin column, which bound BSA and other proteins in the

sample, leaving no heparin binding sites available for sAPRIL, I next sought to isolate sAPRIL

using gel filtration chromatography. Sample in 0.01 M phosphate buffer, pH 7.4, containing 0.14

M NaCl was loaded into a Superdex 200 Increase 10/300 GL column as described in Materials in

Methods. As shown in Figure 8, SYPRO Ruby staining of fraction samples run on SDS-PAGE showed FBS was separated in C1 sample but not FLAG-sAPRIL (Figure 10A. Western blotting showed original sample has FLAG-sAPRIL (Figure 10B).

Figure 10. Purification of sAPRIL by gel filtration chromatography. Samples were analyzed by (A). SYPRO Ruby staining and (B) western blotting using an antibody to APRIL. The input was 5 ml of FLAG-sAPRIL-containing media concentrated to 200 ul as described in Materials and Methods. The numbers above the figure represent the fraction numbers collected from the gel filtration column: 1 ml fractions were collected and thus, e.g., 3 represents the 3rd fraction and 3rd ml collected from the column

3.2.1.4 Purification of FLAG-tagged sAPRIL from HEK 293 cells by ammonium sulfate precipitation

As with DEAE column chromatography, gel filtration chromatography was unable to separate

sAPRIL from BSA in the sample media. I then tried to isolate sAPRIL using ammonium sulfate

precipitation. Precipitates from different concentrations of ammonium sulfate were analyzed by

SDS-PAGE and SYPRO Ruby staining. Figure 11A shows a faint 16 kDa band at the 60%

ammonium sulfate immunoprecipitate. Western blotting using sAPRIL antibody (Figure 11B)

shows that the 16kDa band corresponds to sAPRIL which was detected in the 60% and 70%

ammonium sulfate precipitates. However, BSA was also mostly observed in the 60% and 70%

ammonium sulfate precipitates. Thus, ammonium sulfate precipitation was likewise unable to

separate sAPRIL from BSA in the sample.

Figure 11. Isolation of sAPRIL by ammonium sulfate precipitation. Culture media containing FLAG-tagged sAPRIL was subjected to ammonium sulfate precipitation as described in Materials and Methods. Samples were collected and run on SDS-PAGE for (A) SYPRO Ruby staining and (B) western blot analysis using an APRIL antibody. Data shown represent one of three sets from different experiments.

3.2.2 Purification of GST-tagged sAPRIL by glutathione affinity column chromatography

In a further attempt to obtain purified sAPRIL, I sought to utilize a bacterial expression system to

generate GST-sAPRIL. E.coli (BL 21) was transformed as described in Materials and Methods.

Expressed GST-sAPRIL was isolated from bacterial lysate supernatant using a glutathione-agarose

column, and eluted using reduced glutathione. Column fractions were then analyzed by SDS-

PAGE and Coomassie staining. As shown in Figure 12, GST-sAPRIL, with an expected molecular size of 42 kDa, was expressed in E. coli following a 5 hr induction with IPTG (lane 4). The 42 kDa GST-sAPRIL was isolated from the E. coli lysate supernatant that was loaded into the glutathione agarose column (Figure 12, lanes 9 and 10: E1 and E2). However, the calculated yield

from the supernatant was only 0.1%. Analysis of the lysate pellet (Figure 12, lanes 13 and 23)

showed that the majority of the GST-sAPRIL was in this fraction, indicating localization of the expressed protein in inclusion bodies. To isolate GST-sAPRIL from the inclusion bodies, the

pellet was treated with 0.05% or 0.075% SDS. However, as shown in lanes 18, and 27 and 29, a

minimal amount of GST-sAPRIL was obtained from the SDS-treated bacterial lysate pellet,

indicating that 0.05% and 0.075% SDS was not sufficient to release GST-sAPRIL from the

inclusion bodies. Nonetheless, I obtained sufficient amount of GST-sAPRIL from the bacterial

lysate supernatant to pursue my other experimental aims.

Figure 12. Purification of GST-sAPRIL by glutathione affinity column. GST-sAPRIL expression in E. coli BL21 was induced by IPTG for 5 hrs at 27C. Column samples were analyzed by SDS-PAGE and Coomassie brilliant blue staining. * denotes the isolated GST-sAPRIL; ** denotes the usual heat shock protein contaminant in GST fusion proteins. sAPRIL yield from the cell supernatant fraction was calculated to be 0.1%.

3.3 To identify and characterize the targets of APRIL in breast cancer cells

3.3.1 To identify the targets of APRIL in breast cancer cells.

To identify APRIL binding proteins in T47D breast cancer cells, I used three approaches: (a).

affinity binding to FLAG-sAPRIL immobilized with anti-FLAG agarose, (b). far western blotting

and (c). affinity binding to GST-sAPRIL immobilized with GSH-agarose.

3.3.1.1 Identification of APRIL-interacting proteins by affinity binding to FLAG-sAPRIL immobilized with anti-FLAG agarose

Affinity binding of APRIL interacting proteins in T47D breast cancer cells to FLAG-sAPRIL

immobilized with mouse anti-FLAG agarose was allowed to proceed as described in Materials and

Methods. As shown in Figure 13A (lanes 11 and 12), few proteins eluted from the FLAG- sAPRIL/FLAG-agarose complex at 300mM NaCl. To examine the possibility that the observed bands represent the sAPRIL or the light and heavy chains of the FLAG antibody, the eluted samples were analyzed by western blotting using APRIL and FLAG antibodies. As shown in

Figure 13B, the fractions containing the 21 kDa and 47 kDa proteins (from lanes 11 and 12 in

Figure 13A) did not show immunoreactivity to both the APRIL (Figure 13B, left panel) and FLAG

(Figure 13B, right panel) antibodies. On the other hand, purified FLAG-sAPRIL showed immunoreactivity to both the APRIL and FLAG antibodies while neutrophils showed immunoreactivity to the APRIL antibody. The fact that the secondary goat anti-mouse antibody used in western blotting did not react with the 21 kDa and 57 kDa proteins rules out the possibility that the 2 isolated proteins are the heavy and light chains of the FLAG antibody generated in

mouse. Thus, my results indicate that the 21 kDa and 57 kDa proteins do not correspond to

sAPRIL or the light and heavy chains of FLAG but are s-APRIL binding proteins from the T47D

breast cancer cells The protein band that migrated around the 63 kDa molecular weight standard

(Figure 11A) and was present in the original sample and did not bind to the FLAG-sAPRIL/FLAG- agarose complex as indicated by its elution in the flowthrough, is most likely, BSA from the sample media. This separation of FLAG-sAPRIL from BSA using the FLAG-agarose column is consistent with my result in Figure 6. However, as indicated above, this method of sAPRIL isolation has the limitation of having a very low yield.

To identify the T47D sAPRIL binding proteins isolated using the FLAG-sAPRIL/FLAG-agarose complex, the SDS-PAGE isolated protein bands were cut and re-resolved by SDS-PAGE. A band of proteins that entered within 1 cm of the separating gel was cut and sent for mass spectrometry analysis. As shown in Table 2, 8 putative APRIL-interacting proteins in T47D breast cancer cells, including colony stimulating factor 2 receptor beta common subunit (CSF2RB), a putative 2.2 kDa protein and mutant beta actin were identified.

Figure 13. Isolation of APRIL-interacting proteins in breast cancer cells by affinity binding to FLAG-sAPRIL/FLAG agarose. APRIL interacting proteins in T47D breast cancer cells were allowed to bind to FLAG-sAPRIL/FLAG agarose and isolated as described in Materials and Methods. (A) Coomassie staining of samples loaded and eluted from the FLAG-sAPRIL/FLAG agarose complex. (B). Fractions of interest (300 mM NaCl eluates, corresponding to lanes 11 and 12 in A) were subjected to western blotting using antibodies to APRIL (left panel) and FLAG (right panel). Purified FLAG-sAPRIL and neutrophils were used as positive controls for the blots.

Table 2. APRIL-interacting proteins detected by mass spectrometry of T47D proteins that bind to FLAG-sAPRIL immobilized with anti-FLAG-agarose.

ACCESSION SCORE MASS MATCHES DESCRIPTION No# 1 gi|7959223 45 150746 1 KIAA1481 protein [Homo sapiens] 2 gi|4507599 39 27587 1 Tumor necrosis factor ligand superfamily member 13 [Homo sapiens] 3 gi|4504837 37 45279 1 ATP-sensitive inward rectifier potassium channel 1 isoform 4 gi|119587144 37 17986 1 hCG2040526, isoform CRA_b [Homo sapiens] 5 gi|28592 36 71316 1 serum albumin [Homo sapiens] 6 gi|440575811 34 11752 1 protein CSF2RB [Homo sapiens] 7 gi|28336 32 42128 1 mutant beta-actin (beta'-actin) [Homo sapiens] 8 gi|553734 32 2269 1 putative [Homo sapiens]

3.3.1.2 Identification of APRIL interacting proteins by far western blotting

As an alternative method to identify APRIL-interacting proteins in breast cancer cells, I used far western blotting as described in Materials and Methods. Briefly, T47D breast cancer cell lysate was used as prey and resolved by SDS-PAGE. T47D proteins were transferred onto a PVDF membrane, which was treated to induce the transferred proteins to return to their native structure.

The membrane was then incubated with sAPRIL that was used as bait, followed by western blotting using an antibody to APRIL. As control, I used T47D lysate that was not incubated with the sAPRIL bait. As shown in figure 14, a few APRIL immunoreactive bands (13, 30, 42, 100, and 240 kDa) were detected in the membrane that was exposed to the sAPRIL bait. The potential sAPRIL interacting proteins were isolated by cutting the immunoreactive bands and re-resolving by SDS -PAGE. Once the band of proteins entered the separating gel by about 1 cm, the band was cut and sent for mass spectrometry. As shown in Table 3, around 80 APRIL-interacting candidates were identified from far western blotting. Some of these identified proteins have molecular sizes close to the observed bands in the far western blot (Figure 12) such as CSF2RB (97 kDa), protein

S100-A9 (13.2 kDa), ras-related protein Rab-11A isoform 1 (24.4 kDa), ras-related protein Ral-B

(23.5 kDa), voltage-dependent anion channel 2 (35 kDa), and complement component C3 (188.5 kDa). A putative 2.2 kDa protein was also identified as an APRIL-interacting protein. This putative 2.2 kDa protein as well as CSF2RB were also identified by mass spectrometry as sAPRIL binding proteins from the FLAG-sAPRIL/FLAG-agarose affinity binding approach.

Figure 14. Far western blotting to identify sAPRIL binding proteins. Far western blotting was performed as described in Materials and Methods. T47D lysate was used as prey and sAPRIL (Adipogen) was used as bait. The PVDF membrane was immunoblotted using an antibody to APRIL. T47D lysate that was not exposed to the sAPRIL bait was used as control. * denotes the bands that were cut and analyzed by mass spectrometry.

Table 3. APRIL-interacting proteins detected by mass spectrometry of T47D proteins that bind to FLAG-sAPRIL by far western blotting ACCESSION SCORE MASS MATCHES DESCRIPTION NO#

1 gi|158261511 207 49828 8 unnamed protein product [Homo sapiens] 2 gi|32189394 1338 56525 33 ATP synthase subunit beta, mitochondrial precursor [Homo sapiens] 3 gi|31645 536 36202 12 glyceraldehyde-3-phosphate dehydrogenase [Homo sapiens] 4 gi|28592 413 71316 11 serum albumin [Homo sapiens] 5 gi|4758984 387 24492 16 ras-related protein Rab-11A isoform 1 [Homo sapiens] 6 gi|2506380 333 43894 6 RecName: Full=Lactadherin; AltName: Full=Breast epithelial antigen BA46; AltName: Full=HMFG; AltName: Full=MFGM; AltName: Full=Milk fat globule-EGF factor 8; Short=MFG- E8; AltName: Full=SED1; Contains: RecName: Full=Lactadherin short form 7 gi|34740335 326 50804 9 tubulin alpha-1B chain [Mus musculus] 8 gi|10880989 314 23248 8 ras-related protein Rab-18 isoform 1 [Homo sapiens] 9 gi|119620329 90 28471 4 RAB1A, member RAS oncogene family, isoform CRA_f 10 gi|238427 306 30737 8 Porin 31HM [human, skeletal muscle membranes, Peptide, 282 aa] 11 gi|119574954 288 35029 8 voltage-dependent anion channel 2, isoform CRA_a [Homo sapiens] 12 gi|338695 296 50240 8 beta-tubulin [Homo sapiens] 13 gi|1881449 289 8919 7 hP47 protein [Homo sapiens] 14 gi|1806040 288 48299 9 adipophilin [Homo sapiens] 15 gi|28614 279 39706 11 aldolase A [Homo sapiens] 16 gi|306820 252 27127 12 glutathione transferase M3 [Homo sapiens] 17 gi|5031857 251 36950 7 L-lactate dehydrogenase A chain isoform 1 [Homo sapiens] 18 gi|178849 235 36302 8 apolipoprotein E [Homo sapiens] 19 gi|4506667 234 34423 7 60S acidic ribosomal protein P0 [Homo sapiens] 20 gi|227994 227 14774 8 fatty acid-binding protein 21 gi|5803187 221 37688 8 transaldolase [Homo sapiens] 22 gi|4504067 220 46447 8 aspartate aminotransferase, cytoplasmic [Homo sapiens] 23 gi|4757756 209 38808 6 annexin A2 isoform 2 [Homo sapiens] 24 gi|551638 208 32163 5 SSR alpha subunit [Homo sapiens]

ACCESSION SCORE MASS MATCHES DESCRIPTION NO#

25 gi|2906146 202 35965 4 malate dehydrogenase precursor [Homo sapiens] 26 gi|178757 198 45353 5 apolipoprotein A-IV precursor [Homo sapiens] 27 gi|16751921 197 11391 5 dermcidin isoform 1 preproprotein [Homo sapiens] 28 gi|4557727 182 53699 6 lipoprotein lipase precursor [Homo sapiens] 29 gi|4757810 181 59828 4 ATP synthase subunit alpha, mitochondrial a precursor [Homo sapiens] 30 gi|4504517 179 22826 6 heat shock protein beta-1 [Homo sapiens] 31 gi|28336 171 42128 5 mutant beta-actin (beta'-actin) [Homo sapiens] 32 gi|32097 169 11317 5 unnamed protein product [Homo sapiens] 33 gi|11968009 166 52337 3 nucleotide exchange factor SIL1 precursor [Homo sapiens] 34 gi|809185 164 35840 5 Chain A, The Effect Of Metal Binding On The Structure Of Annexin V And Implications For Membrane Binding 35 gi|553734 150 2269 21 putative [Homo sapiens] 36 gi|11345462 141 20358 4 signal peptidase complex subunit 3 [Homo sapiens] 37 gi|32486 135 35739 3 unnamed protein product [Homo sapiens] 38 gi|4503143 133 45037 4 cathepsin D preproprotein [Homo sapiens] 39 gi|4505753 122 28900 3 phosphoglycerate mutase 1 [Homo sapiens] 40 gi|6970062 122 14858 5 gastric-associated differentially-expressed protein YA6 41 gi|2754812 117 34601 4 Pig3 [Homo sapiens] 42 gi|508285 109 23781 4 Rab5c-like protein, similar to Canis familiaris Rab5c protein, PIR Accession Number S38625 [Homo sapiens] 43 gi|551547 108 25950 4 prosome beta-subunit [Homo sapiens] 44 gi|1065361 107 20610 3 Chain A, Human Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length Non- Myristoylated 45 gi|1673514 98 23621 2 B-cell receptor associated protein, partial [Homo sapiens] 46 gi|119572363 91 30319 2 hCG22067 [Homo sapiens] 47 gi|29424 85 44538 4 beta-1,4-galactosyltransferase (AA -77 to 323) [Homo sapiens] 48 gi|187224 83 37924 2 lactoperoxidase, partial [Homo sapiens] 49 gi|564065 83 36314 2 peroxisomal enoyl-CoA hydratase-like protein [Homo sapiens] 50 gi|14248539 82 79982 2 stonin 2 [Homo sapiens]

ACCESSION SCORE MASS MATCHES DESCRIPTION NO#

51 gi|1633054 73 18098 2 Chain A, Cyclophilin A Complexed With Dipeptide Gly-Pro 52 gi|179665 70 188585 3 complement component C3 [Homo sapiens] 53 gi|6912486 64 15511 2 U6 snRNA-associated Sm-like protein LSm4 isoform 1 54 gi|1326083 61 59537 4 butyrophilin precursor [Homo sapiens] 56 gi|13325075 58 83324 1 sulfhydryl oxidase 1 isoform a precursor [Homo sapiens] 57 gi|984267 52 148624 1 xanthine dehydrogenase [Homo sapiens] 58 gi|119594981 51 15779 2 hCG2016955, partial [Homo sapiens] 59 gi|1495421 51 31020 2 mono-ADP-ribosyltransferase, partial [Homo sapiens] 60 gi|2065179 51 22383 1 unnamed protein product [Homo sapiens] 61 gi|5174723 50 38211 2 mitochondrial import receptor subunit TOM40 homolog 62 gi|440575811 48 11752 7 protein CSF2RB [Homo sapiens] 63 gi|4506405 48 23508 1 ras-related protein Ral-B [Homo sapiens] 64 gi|6688197 48 33743 1 PAP-inositol-1,4-phosphatase [Homo sapiens] 65 gi|12804561 48 14884 2 Ribosomal protein S15a [Homo sapiens] 66 gi|7572969 45 8450 2 immunoglobulin heavy chain variable region [Homo sapiens] 67 gi|6478782 42 39556 2 long-chain L-2-hydroxy acid oxidase [Homo sapiens] 68 gi|180576 41 43247 2 creatine kinase M [Homo sapiens] 69 gi|4049585 41 173888 2 Slit-1 protein [Homo sapiens] 70 gi|4506773 40 13291 1 protein S100-A9 [Homo sapiens] 71 gi|34534527 40 86904 2 unnamed protein product [Homo sapiens] 72 gi|180117 38 53588 1 antigen CD36 [Homo sapiens] 73 gi|7329718 35 257758 1 hypothetical protein [Homo sapiens] 74 gi|13477197 35 31152 1 Quinolinate phosphoribosyltransferase [Homo sapiens] 75 gi|22209028 31 32185 1 Thioredoxin-related transmembrane protein 1 [Homo sapiens] 76 gi|29726390 30 25211 1 Chain A, Molecular Basis For The Local Confomational Rearrangement Of Human Phosphoserine Phosphatase 77 gi|251840 30 110118 1 glutamate receptor subunit [Homo sapiens] 78 gi|29888 29 10988 1 unnamed protein product [Homo sapiens] 79 gi|9844110 29 23995 1 cAMP-specific phosphodiesterase 4D [Homo sapiens]

ACCESSION SCORE MASS MATCHES DESCRIPTION NO#

80 gi|179462 28 64720 1 N-acetyl-beta-glucosaminidase prepro- polypeptide, partial [Homo sapiens]

3.3.1.3 Identification of APRIL interacting proteins by affinity binding to GST-sAPRIL

immobilized with glutathione agarose

Another approach that I used to identify APRIL-interacting proteins in T47D cells was by affinity

binding to GST-sAPRIL attached to GSH- agarose. The procedure described in Materials and

Methods was followed. For this study, GST-empty vector (from a lysate of E.coli transformed with an empty GST vector) attached to GSH- agarose was used as control affinity matrix.

Following incubation with T47D cell lysates, reduced GST eluates from the GST-sAPRIL/GSH- agarose and GST-empty vector/GSH-agarose columns were sent for mass spectrometry Table 4 shows a list of sAPRIL interacting proteins that were identified in the GST-sAPRIL/GSH-agarose eluate. Proteins that were identified in both the GST-sAPRIL/GSH-agarose and GST-empty vector/GSH-agarose eluates were not included in the list. The identified sAPRIL interacting proteins include a putative 2.2 kDa protein, dynein light chain 1, cytoplasmic, BCSG1 protein, chain A and macrophage migration inhibitory factor (Mif). Thus, the putative 2.2 kDa protein was identified as an sAPRIL interacting protein by affinity binding to FLAG-sAPRIL and GST- sAPRIL and by far western blotting. On the other hand, the CSF2RB was identified as an sAPRIL interacting protein by affinity binding to FLAG-sAPRIL and by far western blotting. Interestingly, by multiple sequence alignment using T-coffee and database search using Blast, I found that the

CSF2RB ligand, colony stimulating factor 2 (CSF2 or granulocyte macrophage colony stimulating factor, GM-CSF), has 41% similarity to APRIL and, CSF2RB has 25% similarity to each of the

known immune cell APRIL receptors, BCMA and TACI (Figure 15). In addition, the putative 2.2 kDa APRIL-interacting protein has 100% homology to residues 30 to 48 and 31 to 50 of the T cell

receptor α and β isoform, respectively (Figure 16). The 2.2 kDa APRIL-interacting protein also has some similarity to BCMA, TACI and CSF2RB but these were not significant.

Table 4. APRIL-interacting proteins detected by mass spectrometry of T47D proteins

that bind to GST-sAPRIL immobilized with glutathione agarose. A: Proteins bound to

glutathione agarose; B: Proteins bound to GST-sAPRIL immobilized with glutathione

agarose.

ACCESSION SCORE MASS MATCHES DESCRIPTION NO# 1 gi|4502599 290 30641 7 carbonyl reductase [NADPH] 1 isoform 1 [Homo sapiens 2 gi|32111 201 14164 8 unnamed protein product [Homo sapiens] 3 gi|32097 153 11317 7 unnamed protein product [Homo sapiens] 4 gi|28592 90 71316 3 serum albumin [Homo sapiens] 5 gi|178024 80 7908 2 beta-actin, partial [Homo sapiens] 6 gi|306820 72 27127 3 glutathione transferase M3 [Homo sapiens] 7 gi|38051823 64 93263 3 Plasminogen [Homo sapiens] 8 gi|467828 54 17772 2 smooth muscle myosin alkali light chain [Homo sapiens] 9 gi|5174445 53 45995 2 lanC-like protein 1 [Homo sapiens] 10 gi|24308179 46 70571 2 CTTNBP2 N-terminal-like protein [Homo sapiens] 11 gi|386772 35 15352 1 histone H3, partial [Homo sapiens]

ACCESSION SCORE MASS MATCHES DESCRIPTION NO# 1 gi|4502599 224 30641 5 carbonyl reductase [NADPH] 1 isoform 1 [Homo sapiens 2 gi|4826898 117 15216 4 profilin-1 [Homo sapiens] 3 gi|28592 109 71316 4 serum albumin [Homo sapiens] 4 gi|5032057 101 11847 5 protein S100-A11 [Homo sapiens] 5 gi|32097 80 11317 4 unnamed protein product [Homo sapiens] 6 gi|8100054 72 10244 2 dolichol-phosphate-mannose synthase [Homo sapiens] 7 gi|3659901 70 11379 2 F1F0-type ATP synthase subunit g [Homo sapiens] 8 gi|4503029 67 15854 3 cellular retinoic acid-binding protein 2 [Homo sapiens]

ACCESSION SCORE MASS MATCHES DESCRIPTION NO# 9 gi|32111 62 14164 2 unnamed protein product [Homo sapiens] 10 gi|4008131 61 10576 1 chaperonin 10 [Homo sapiens] 11 gi|5802976 61 7850 1 adipogenesis regulatory factor [Homo sapiens] 12 gi|306820 59 27127 2 glutathione transferase M3 [Homo sapiens] 13 gi|1942977 53 12612 1 Chain A, Macrophage Migration Inhibitory Factor 14 gi|4503253 47 12660 1 dolichyl-diphosphooligosaccharide--protein glycosyltran 15 gi|106258 42 41135 3 glycoprotein-fucosylgalactoside alpha-N- acetylgalactosa 16 gi|4505813 42 10530 1 dynein light chain 1, cytoplasmic [Homo sapiens] 17 gi|553734 41 2269 2 putative [Homo sapiens] 18 gi|14249376 40 6510 1 up-regulated during skeletal muscle growth protein 5[Homo sapiens] 19 gi|2281474 40 13321 1 BCSG1 protein [Homo sapiens] 20 gi|28336 38 42128 1 mutant beta-actin (beta'-actin) [Homo sapiens] 21 gi|4506711 36 9797 1 40S ribosomal protein S27 [Homo sapiens] 22 gi|340062 36 17776 1 pro-ubiquitin, partial [Homo sapiens] 23 gi|38051823 35 93263 1 Plasminogen [Homo sapiens] 24 gi|11128019 31 11855 1 cytochrome c [Homo sapiens] 25 gi|4505357 31 9421 1 cytochrome c oxidase subunit NDUFA4 [Homo sapiens]

A. Sequence alignment of CSF2 and APRIL

CSF2 ------MWLQSLLLLGTVACSISAPARSP------APRIL MPASSPFLLAPKGPPGNMGGPVREPALSVALWLSWGAALGAVACAMALLTQQTELQSLRR :**. **:***::: ::.

CSF2 ------SPSTQPWEHVN------AIQEARRLLNLSRDTAAEM--- APRIL EVSRLQGTGGPSQNGEGYPWQSLPEQSSDALEAWENGERSRKRRAVLTQKQKKQHSVLHL . . **: : :: * :*. .:.. .:

CSF2 ---N----ETVEVISEMFDLQEPTCLQTRLELYKQGLR------GSLTK APRIL VPINATSKDDSDVTEVMWQ----PALRRGRGLQAQGYGVRIQDAGVYLLYSQVLFQDVTF * : :* . *:: .*: * ** .:*

CSF2 LKGPLTMMASH------YKQHCPPTPE---TSCATQIITFESFKENLKDFLLVIPFDC APRIL TMGQVVSREGQGRQETLFRCIRSMPSHPDRAYNSCYSA----GVFHLHQGDILSVIIPRA * :. .: : * *: .** : *: . *:* ** .

CSF2 WEPVQ------E- APRIL RAKLNLSPHGTFLGFVKL :: :

B. Sequence alignment of CSF2RB and BCMA

CSF2RB MVLAQGLLSMALLALCWERSLAGAEETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVT BCMA ------MLQM--AGQCSQ------NEYFDS------:*.* . * : *:* .

CSF2RB LIRRVNEDLLEPVSCDLSDDMPWSACPHPRCVPRRCVIPCQSFVVTDVDYFSFQPD---- BCMA ------LLHACIPCQ------LRCSSNTPPLTCQRYCNASVTNSVKGTNAILW * : *: ** . : ** : :.* :

CSF2RB RPLGTRLTVTLTQHVQPPEPRDLQISTDQDHFLLTWSVALGS------PQSHWLSPG BCMA TCLGLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPR ** * ::*: .* *.:: . :*.* * * ** .. : *

CSF2RB DLEFEVVYKRLQDSWEDAAILLSNTSQATLGPEHLMPSSTYVARVRTRLAPGSRLSGRPS BCMA GLEYTVEE------**: *

CSF2RB KWSPEVCWDSQPGDEAQPQNLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPDAGEE BCMA -CTCEDCIKSK------PKVDSD : * * .*: *.. .:

CSF2RB ECSPVLREGLGSLHTRHHCQIPVPDPATHGQYIVSVQPRRAEKHIKSSVNIQMAPPSLNV BCMA HCFP------.* *

CSF2RB TKDGDSYSLRWETMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPSTR BCMA ------LPAMEEGAT ***:* .:

CSF2RB YWARVRVRTSRTGYNGIWSEWSEARSWDTESVLPMWVLALIVIFLTIAVLLALRFCGIYG BCMA IL------

CSF2RB YRLRRKWEEKIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPV BCMA ------

CSF2RB GFGDSEVSPLTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSF BCMA ------VTTKTNDYCK------. . :. *.

CSF2RB DFNGPYLGPPHSRSLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMG BCMA ------SLPAALSATEIEKSISAR------*** *. * :. .::

CSF2RB PGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGV BCMA ------

CSF2RB ASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYV BCMA ------

CSF2RB ELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPG BCMA ------

CSF2RB LGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPP BCMA ------

CSF2RB WEVNKPGEVC BCMA ------

C. Sequence alignment of CSF2Rb and TACI

CSF2RB MVLAQGLLSMALLALCWERSLAGAEETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVT TACI ------

CSF2RB LIRRVNEDLLEPVSCDLSDDMPWSACPHPRCVPRRCVIPCQSFVVTDVDYFSFQPDRPLG TACI ------

CSF2RB TRLTVTLTQHVQPPEPRDLQISTDQDHFLLTWSVALGSPQSHWLSPGDLEFEVVYKRLQD TACI ------

CSF2RB SWEDAAILLSNTSQATLGPEHLMPSSTYVARVRTRLAPGSRLSGRP-SKWSPEVCWDSQP TACI ------MSGLGRSRRGGRSR------VDQEERFPQGL-WTGVAMRSCPEEQYWDPLL :* .:: * . . . *: * :* . * **

CSF2RB GDEAQPQNLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPDAGEEECSPVLREGLGS TACI GTCMSCKTI-CNH--QSQRTCAAF-CRSLSCRKEQGKFYDHL------LRDCI-- * . :.: * . : :*: :.::. . * **. **: :

CSF2RB LHTRHHCQIPVPDPATHGQYIVSVQPRRAEKHIKSSVNIQMAPPSLNVTKDGDSYSLRWE TACI -----SCASICGQHPKQCAYFC------ENKLRSPVNL---PPELRRQRSGEVE----- * : .: *: *::::* **: **.*. :.*:

CSF2RB TMKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPSTRYWARVRVRTSRT TACI ------NNSDNSG . .

CSF2RB GYNGIWSEWSEARSWDTESVLPMWVL---ALIVIFLTIAVLLALRFCGIYGYRLRRKWEE TACI RYQGLEHRGSE-----ASPALPGLKLSADQVALVYSTLGLCLCAVLCCFLVAV------*:*: . ** :. .** * : ::: *:.: *. :* :

CSF2RB KIPNPSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSP TACI ------ACF ..

CSF2RB LTIEDPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGP TACI LKKRGDPCSCQPRSRPRQSPAKSSQDH----AMEAGSPVSTSPEPVETCSFCFPEC--RA *. . *:* * * :** *. : : * *.:: ::.** *

CSF2RB PHSRSLPDILGQPEPPQEGGS------QKSPPPGSLEYLCLPAGGQVQLVPLAQA TACI PTQES-AVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQ------E * ..* * *:* * * ..* :*:**

CSF2RB MGPGQAVEVERRPSQGAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDP TACI GGPGA------***

CSF2RB GVASGYVSSADLVFTPNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEG TACI ------

CSF2RB YVELPPIEGRSPRSPRNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFL TACI ------

CSF2RB PGLGPGPLSLRSKPSSPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSL TACI ------

CSF2RB PPWEVNKPGEVC TACI ------

Figure 15. Amino acid sequence alignment of (A) CSF2 and APRIL, (B) CSF2RB and

BCMA and, (C) CSF2RB and TACI. T-Coffee and BLAST analyses revealed that the

CSF2RB ligand, CSF2, has 41% similarity to APRIL, and CSF2RB has 25% similarity to both

BCMA and TACI. Below the protein sequences, (*), (:) and (.) denote conserved sequence, conservative mutation and semi-conservative mutation, respectively.

A. Sequence alignment of the 2.2 kDa putative protein and T cell receptor  and 

TCRα ATLTKKESFLHITAPKPEDSATYLCAVR--ETGANSKLTFGKGITLSVRP TCRβ FQKARKSANLVISASQLGDSAMYFCAMREGYTGANSKLTFGKGITLSVRP 2.2kDa putative ------CYTGANSKLTFGKGITLSVRP *******************

TCRa DIQNPDPAVYQLRDSKSSDKSVCL TCRb DIQNPDPAVYQLRDSKSSDKSVCL 2.2kDa putative ------

B. Sequence alignment of the 2.2 kDa putative protein and BCMA

putative ------CY------TGANSKL---- BCMA MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRYCNASVTNSVKGTNAILWTCL * .*:*: *

putative ------TFGKGIT BCMA GLSLIISLAVFVLMFLLRKINSEPLKDEFKNTGSGLLGMANIDLEKSRTGDEIILPRGLE : :*:

putative LSVRP------BCMA YTVEECTCEDCIKSKPKVDSDHCFPLPAMEEGATILVTTKTNDYCKSLPAALSATEIEKS :*.

putative, ---- BCMA ISAR

C. Sequence alignment of the 2.2 kDa putative protein and TACI

Putative ------TACI TCAAFCRSLSCRKEQGKFYDHLLRDCISCASICGQHPKQCAYFCENKLRSPVNLPPELRR

Putative ------CYTG TACI QRSGEVENNSDNSGRYQGLEHRGSEASPALPGLKLSADQVALVYSTLGLCLCAVLCCFLV *:

putative ANSKLTFGKGITLSVRP------TACI AVACFLKKRGDPCSCQPRSRPRQSPAKSSQDHAMEAGSPVSTSPEPVETCSFCFPECRAP * : : :* * :*

putative ------TACI TQESAVTPGTPDPTCAGRWGCHTRTTVLQPCPHIPDSGLGIVCVPAQEGGPGA

D. Sequence alignment of the 2.2 kDa putative protein and CSF2RB

CSF2RB MVLAQGLLSMALLALCWERSLAGAEETIPLQTLRCYNDYTSHITCRWADTQDAQRLVNVT putative ------CYTGANSKLTFG------**. .*::*

CSF2RB LIRRVNEDLLEPVSCDLSDDMPWSACPHPRCVPRRCVIPCQSFVVTDVDYFSFQPDRPLG putative ------

CSF2RB TRLTVTLTQHVQPPEPRDLQISTDQDHFLLTWSVALGSPQSHWLSPGDLEFEVVYKRLQD putative K------

CSF2RB SWEDAAILLSNTSQATLGPEHLMPSSTYVARVRTRLAPGSRLSGRPSKWSPEVCWDSQPG putative ------GITLSVRP------* ** **

CSF2RB DEAQPQNLECFFDGAAVLSCSWEVRKEVASSVSFGLFYKPSPDAGEEECSPVLREGLGSL Putative ------

CSF2RB HTRHHCQIPVPDPATHGQYIVSVQPRRAEKHIKSSVNIQMAPPSLNVTKDGDSYSLRWET putative ------

CSF2RB MKMRYEHIDHTFEIQYRKDTATWKDSKTETLQNAHSMALPALEPSTRYWARVRVRTSRTG putative ------

CSF2RB YNGIWSEWSEARSWDTESVLPMWVLALIVIFLTIAVLLALRFCGIYGYRLRRKWEEKIPN putative ------

CSF2RB PSKSHLFQNGSAELWPPGSMSAFTSGSPPHQGPWGSRFPELEGVFPVGFGDSEVSPLTIE putative ------

CSF2RB DPKHVCDPPSGPDTTPAASDLPTEQPPSPQPGPPAASHTPEKQASSFDFNGPYLGPPHSR putative ------

CSF2RB SLPDILGQPEPPQEGGSQKSPPPGSLEYLCLPAGGQVQLVPLAQAMGPGQAVEVERRPSQ putative ------

CSF2RB GAAGSPSLESGGGPAPPALGPRVGGQDQKDSPVAIPMSSGDTEDPGVASGYVSSADLVFT putative ------

CSF2RB PNSGASSVSLVPSLGLPSDQTPSLCPGLASGPPGAPGPVKSGFEGYVELPPIEGRSPRSP putative, ------

CSF2RB RNNPVPPEAKSPVLNPGERPADVSPTSPQPEGLLVLQQVGDYCFLPGLGPGPLSLRSKPS putative, ------

CSF2RB SPGPGPEIKNLDQAFQVKKPPGQAVPQVPVIQLFKALKQQDYLSLPPWEVNKPGEVC putative, ------

Figure 16. Amino acid sequence alignment of the 2.2 kDa putative APRIL-interacting protein and (A) T cell receptor α and β, (B) BCMA, (C) TACI and (D) CSF2RB. T-coffee and Blast analyses revealed that the putative APRIL-interacting protein has 100% homology to aa 30-48 and 31-50 of the T cell receptor α and β isoform, respectively, but has insignificant

similarity to BCMA, TACI and CSF2RB. (*), (:) and (.) indicate conserved sequence, conservative mutation, and semi-conservative mutation, respectively.

3.3.2 Classification of identified APRIL interacting proteins in T47D cells

In Table 5, I classify the APRIL interacting proteins identified by mass spectrometry following three approaches: affinity binding to FLAG-sAPRIL, affinity binding to GST-sAPRIL and far western blotting. The identified proteins were classified into 31 groups based on function.

Interestingly, several of these proteins play a role in cell proliferation, survival and apoptosis.

Table 5. Classification of APRIL-binding proteins based on cell function.

FUNCTION PROTEINS

TRANSCRIPTIONAL • Histone H2A type 1-B/E REGULATION,CHROMOSO • Adipogenesis regulatory factor MAL INSTABILITY DNA REPLICATION , DNA • Human H4 histone REPAIR, DNA DAMAGE • Mono-ADP-ribosyltransferase, partial • Prosome beta-subunit RNA TRANSLATION • 40S ribosomal protein S27 • Ribosomal protein S15a

CELL CYCLE • Aldolase A • Ribosomal protein S15a • Beta-tubulin • Prosome beta-subunit • PROTEIN SYNTHESIS • 40S ribosomal protein S27 • Tubulin alpha-1B chain • 60S acidic ribosomal protein P0 • SSR alpha subunit or Signal sequence receptor Subunit alpha • Heat-shock proteins, Hsp84 and Hsp86 • Beta-1,4-galactosyltransferase (AA 77 - 323) • Signal peptidase complex subunit 3 • Sulfhydryl oxidase 1 isoform a precursor • Thioredoxin-related transmembrane protein 1 • B-cell receptor associated protein, partial CELL PROLIFERATION • A proliferation inducing ligand • Chain A, Macrophage Migration Inhibitory Factor (Mif) • Lactadherin; AltName: Full=Breast epithelial antigen BA46 • Aldolase A • Apolipoprotein E • Annexin A2 isoform 2 • Xanthine dehydrogenase • ras-related protein Ral-B • Ribosomal protein S15a • protein CSF2RB CELL GROWTH • protein S100-A9 • PAP-inositol-1,4-phosphatase • Sulfhydryl oxidase 1 isoform a precursor APOPTOSIS • A proliferation inducing ligand • Chaperonin 10 • Dolichyl-diphosphooligosaccharide--protein Glycosyltransferase subunit DAD1

FUNCTION PROTEINS

• Cytochrome c • Glycoprotein-fucosylgalactoside alpha-N- Acetylgalactosaminyltransferase (EC 2.4.1.40) A1 allele [validated] – human • Lactadherin; AltName: Full=Breast epithelial antigen BA46 • mono-ADP-ribosyltransferase, partial • PAP-inositol-1,4-phosphatase • Porin 31HM [human, skeletal muscle membranes, Peptide, 282 aa] • Voltage-dependent anion channel 2, isoform CRA_a • Glutamate receptor subunit • B-cell receptor associated protein, partial • Prosome beta-subunit CELL SURVIVAL • A proliferation inducing ligand • Chain A, Macrophage Migration Inhibitory Factor (Mif) • Dermcidin isoform 1 preproprotein CELL ADHESION • Lactadherin; AltName: Full=Breast epithelial antigen BA46 • RAB1A, member RAS oncogene family, isoform CRA_f Peptide • Apolipoprotein A-IV precursor

METABOLIC FUNCTION • Carbonyl reductase [NADPH] 1 isoform 1 • Dolichol-phosphate-mannose synthase • Maintaining the ATP synthase population in mitochondria • 40S ribosomal protein S27 • Cytochrome c oxidase subunit NDUFA4 • ATP synthase subunit beta, mitochondrial precursor • Glyceraldehyde-3-phosphate dehydrogenase • Tubulin alpha-1B chain • Adipophilin • Aldolase A • Glutathione transferase M3 • L-lactate dehydrogenase A chain isoform 1 • Apolipoprotein E • Transaldolase • Aspartate aminotransferase, cytoplasmic • SSR alpha subunit or Signal sequence receptor subunit alpha • Malate dehydrogenase precursor • Glutamate receptor subunit • Apolipoprotein A-IV precursor • Quinolinate phosphoribosyltransferase • Antigen CD36 or Platelet glycoprotein 4 • Creatine kinase M • Long-chain L-2-hydroxy acid oxidase

FUNCTION PROTEINS

• Mitochondrial import receptor subunit TOM40 • Xanthine dehydrogenase • Biliverdin IX alpha reductase • Peroxisomal enoyl-CoA hydratase-like protein • Phosphoglycerate mutase 1 • Cathepsin D preproprotein • Lipoprotein lipase precursor HOMEOSTASIS • ATP-sensitive inward rectifier potassium channel 1 Isoform • hP47 protein CELL COMMUNICATION • cAMP-specific phosphodiesterase 4D

CELL-CELL INTERACTION • beta-1,4-galactosyltransferase (AA 77- 323) • Protein S100-A9

CELL SIGNALING SIGNAL • BCSG1 protein or Gamma-synuclein. Breast cancer- TRANSDUCTION PATHWAY specific gene 1 protein • ras-related protein Rab-18 isoform 1 • mono-ADP-ribosyltransferase, partial • PAP-inositol-1,4-phosphatase • Protein S100-A9 • cAMP-specific phosphodiesterase 4D • Protein CSF2RB • Butyrophilin precursor • Prosome beta-subunit

CELL SHAPE • KIAA1481 protein /Protein shroom3 • Mutant beta-actin (beta'-actin) • Profilin-1 • Tubulin alpha-1B chain • Aldolase A TRANSPOT • Serum albumin • F1F0-type ATP synthase subunit g • Cytochrome c • Cellular retinoic acid-binding protein 2 • Cytochrome c oxidase subunit NDUFA4 • ATP synthase subunit beta, mitochondrial precursor • ras-related protein Rab-11A isoform 1 • ras-related protein Rab-18 isoform 1 • RAB1A, member RAS oncogene family, isoform CRA_f • Fatty acid-binding protein • Nucleotide exchange factor SIL1 precursor • Rab5c-like protein, similar to Canis familiaris Rab5c • Chain A, Human Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length Non-Myristoylated

FUNCTION PROTEINS

• Porin 31HM • Voltage-dependent anion channel 2, isoform CRA_a • Glutamate receptor subunit [Homo sapiens] • Mitochondrial import receptor subunit TOM40 CELL MOTILITY • Mutant beta-actin (beta'-actin) • Aldolase A • beta-tubulin CELL DIFFERENTIATION • Carbonyl reductase [NADPH] 1 isoform 1 • Protein S100-A11 • Chaperonin 10 • Adipogenesis regulatory factor • Stonin 2 • Xanthine dehydrogenase • 40 13291 1 1 1 1 0.26 protein gi|4506773 Porin 31HM [human, skeletal muscle membranes, • voltage-dependent anion channel 2, isoform CRA_a IMMUNE FUNCTION • Profilin-1 • Glycoprotein-fucosylgalactoside alpha-N- • Apolipoprotein E • Dermcidin isoform 1 preproprotein • Chain A, Cyclophilin A Complexed With Dipeptide Gly-Pro • Complement component C3 • Protein S100-A9 • Porin 31HM [human, skeletal muscle membranes, Peptide, 282 aa] • Antigen CD36 /Platelet glycoprotein 4 • Voltage-dependent anion channel 2, isoform CRA_a • Lactoperoxidase, partial • Prosome beta-subunit • INHBITORY PROTEIN • Profilin-1

CELL MIGRATION • ras-related protein Ral-B • RAB1A, member RAS oncogene family, isoform CRA_f Peptide, CELLULAR RESPONSE • Adipogenesis regulatory factor

FIBRINOLYSIS • Plasminogen

ANGIOGENESIS • RecName: Full=Lactadherin; AltName: Full=Breast epithelial antigen BA46 • Annexin A2 isoform 2 • Antigen CD36 /Platelet glycoprotein 4

FUNCTION PROTEINS

MEMBRANE FUNCTON • Annexin A2 isoform 2 • Rab5c-like protein, similar to Canis familiaris Rab5c protein, PIR Accession Number S38625 • Chain A, Human Adp-Ribosylation Factor 1 Complexed With Gdp, Full Length Non-Myristoylated • Ribosomal protein S15a • Glutamate receptor subunit • ras-related protein Ral-B GENE EXPRESSION • SSR alpha subunit/Signal sequence receptor subunit alpha • Rosome beta-subunit • Mono-ADP-ribosyltransferase, partial • Xanthine dehydrogenase • Signal peptidase complex subunit 3 • ras-related protein Ral-B INVOLVED IN STRESS • Heat shock protein beta-1 RESISTANCE AND ACTIN ORGANIZATION UNKNOWN • Putative 2.269 kDa • hCG22067 gi|119572363 • pro-ubiquitin, partial [Homo sapiens] • Gastric-associated differentially-expressed protein YA61P • 52 1 NCBInr gi|6912486 64 15511 2 2 1 1 0.22 U6 snRNA- associated Sm-like protein LSm4 isoform 1 • Immunoglobulin heavy chain variable region [Homo sapien • Slit-1 protein [Homo sapiens] • Hypothetical protein [Homo sapiens] • Unnamed protein product gi|34534527 • Chain A, Molecular Basis For The Local Confomational Rearrangement Of Human Phosphoserine Phosphatase • N-acetyl-beta-glucosaminidase prepro-polypeptide, partial • Unnamed protein product gi|29888 • hCG2016955, partial gi|119594981

Characterization of APRIL interaction with CSF2RB

Since the CSF2RB ligand, CSF2, has significant homology to APRIL, I sought to further characterize APRIL interaction with CSF2RB. To do so, I initially transfected HEK293 cells with

FLAG-sAPRIL and/or CSF2RB-Myc as described in Materials and Methods.

3.3.2.1 Analysis of sAPRIL and CSF2RB interaction by coimmunoprecipitation

To examine sAPRIL and CSF2RB interaction by coimmunoprecipitation, I first tested whether I can successfully immunoprecipate these proteins using available antibodies. As shown in Figure

17A, right panel (lane 1), I can immunoprecipitate FLAG-sAPRIL from transfected HEK 293 cells using an antibody to APRIL and detect the protein on a western blot using an antibody to FLAG.

Figure 17B, right panel (lane 1), shows that I can immunoprecipitate CSF2RB-Myc from transfected HEK 293 cells using a Myc antibody and detect the protein on a western blot using an antibody to CSF2RB. Specificity of immunoreactivity was validated using control immunoprecipitates with normal mouse IgG (Figure 17A and B, right panels, lane 2), and western blots of non-transfected and transfected cell lysates using APRIL and CSF2RB antibodies (Figure

17 A and B, left panels, lanes 1 and 3).

To determine whether co-transfected FLAG- sAPRIL and CSF2RB-Myc in HEK293 cells interact with each other, cell lysates were subjected to immunoprecipitation using an antibody to APRIL or Myc. Each immunoprecipitate was then immunoblotted with an antibody to FLAG or CSF2RB.

As shown in Figure 18A, lane 3, the Myc immunoprecipitate immunoreacted with the FLAG antibody, indicating coimmunoprecipitation of FLAG-sAPRIL with CSF2RB-Myc. Consistent with this observation, Figure 18B, lane 3, shows that the APRIL immunoprecipitate

immunoreacted with the CSF2RB antibody, indicating coimmunoprecipitation of CSF2RB-Myc with FLAG- sAPRIL. Together, these observations support my mass spectrometry finding that

CSF2RB is an APRIL-interacting protein.

Figure 17. Immunoprecipitation of sAPRIL and CSF2RB expressed in HEK 293 cells. HEK293 cells were transfected with FLAG-sAPRIL or CSF2RB- Myc as described in Materials and Methods. (A) FLAG-sAPRIL and (B) CSF2RB-Myc were immunoprecipitated using antibodies to APRIL and Myc, respectively, and detected in western blots using antibodies to FLAG and CSF2RB, respectively. For immunoprecipitation control, normal mouse IgG was used for immunoprecipitation of transfected cell lysates. The antibody heavy and light chains were detected by the secondary anti-mouse antibody. As positive controls for sAPRIL and CSF2RB detection, lysates of HEK 293 cells transfected with FLAG-sAPRIL or CSF2RB-Myc were immunobotted with an antibody to APRIL or CSF2RB, respectively. As negative controls, non- transfected HEK 293 cells and their culture media were immunoblotted using an antibody to APRIL or CSF2R

Figure18. Coimmunoprecipitation of FLAG-sAPRIL and CSF2RB-Myc. HEK 293 cells were co-transfected with FLAG-sAPRIL and CSF2RB-Myc as described in Materials and Methods. FLAG-sAPRIL and CSF2RB-Myc were immunoprecipitated using antibodies to APRIL and Myc, respectively. The immunoprecipitates and corresponding controls were immunoblotted with antibodies to (A) FLAG and (B) CSF2RB. The immunoblot positive controls were lysates of the HEK 293 co-transfected cells probed with FLAG and CSF2RB antibodies. The negative controls were lysates of non-transfected cells probed with the same antibodies. The light and heavy chains of the immunoprecipitating antibodies were distinguished using control normal mouse IgG immunopreciptates which show both components of the antibody.

3.3.2.2 Analysis of sAPRIL and CSF2RB interaction by far western blotting

To determine whether the interaction between sAPRIL and CSF2RB is direct, lysate of HEK293

cells transfected with CSF2RB-Myc was subjected to immunoprecipitation using anti-myc. The

Myc immunoprecipitate was then resolved by SDS-PAGE and subjected to far western blotting.

Thus, proteins from the CSF2RB-expressing HEK 293 cells served as prey. FLAG-s-APRIL

(Adipogen) served as bait. As shown in the far western blot (Figure 19, top panel), APRIL immunoreactivity was detected (between 75 and 100 kDa) in the Myc immunoprecipitate (last lane), indicating that APRIL interacts with CSF2RB directly. Two non-specific APRIL immunoreactive bands between 63 and 75 kDa were detected in both the FLAG-s-APRIL

(Adipogen; expected migration at 35 kDa) sample and the normal IgG immunoprecipitate from

HEK 293 cells transfected with CSF2RB-Myc.

Figure 19. APRIL interacts with CSF2RB directly. Far western blotting was performed as described in Materials and Methods. The left panel is a blot using APRIL antibody. Samples of non-transfected HEK 293 cell lysate (100 ug, 1st lane), FLAG-sAPRIL (Adipogen; 3 ug, 2nd lane), normal IgG immunoprecipitate (3rd lane) and Myc immunoprecipitate (4th lane) from HEK 293 cells transfected with CSF2RB-Myc were analyzed. The FLAG-sAPRIL sample served as positive control for the APRIL antibody. The non-transfected HEK 293 cell lysate and IgG immunoprecipitate served as negative controls. The right panel shows the normal IgG immunoprecipitate (from the 3rd lane on the left panel) and Myc immunoprecipitate (from the 4th lane on the left panel) lanes reblotted with an antibody to CSF2RB. Autoradiograph exposure was done overnight for the top panel and 5 min for the bottom panel.

3.4 To determine whether pi3k/akt and stat3 are involved in the proposed april-mediated breast cancer cell proliferation.

Previously, it was reported that the PI3K/AKT pathway is involved in APRIL-mediated

proliferation of colon cancer cells. In breast cancer cells as well as in other solid and hematological tumors such as ovarian carcinoma, colorectal carcinoma, renal carcinoma and multiple myeloma, constitutive stimulation of STAT3 was observed (Banerjee and Resat 2016, Levy and Lee 2002,

Gkouveris et al. 2015). Interestingly, both the PI3K/AKT and STAT3 pathways have been

implicated in the signaling of CSF2RB which I have identified as an APRIL-interacting protein.

I, therefore, hypothesized that APRIL induces proliferation of breast cancer cells via the

PI3K/AKT and/or STAT3 pathways. To address this hypothesis, T47D breast cancer cells treated

with sAPRIL at different time points were analyzed for AKT and STAT3 activation. As shown in

Figure 18, STAT3 Tyr705 phosphorylation was detectable in T47D cells prior to sAPRIL

treatment (time 0), indicating constitutive activation of STAT3 as previously described in cancer cells (Banerjee and Resat 2016). However, 5 min following sAPRIL treatment, a noticeable

increase in STAT Tyr705 phosphorylation was observed, indicating enhanced STAT3 activation.

Enhanced STAT3 activation was sustained until the end of analysis at 72 hrs. Similarly, minimal

but detectable phosphorylation of AKT at Tyr308 and Ser473 was detectable in T47D cells prior

to sAPRIL treatment and until 15 min of exposure to sAPRIL. However, considerable increase in

AKT phosphorylation at both Thr308 and Ser473 was noted after 30 min of exposure to sAPRIL.

As with STAT3 Tyr 705, increased Thr308 and Ser473 phosphorylation of AKT was sustained

until the end of analysis at 72 hrs. These observations indicate that both the STAT3 and PI3K/AKT pathways are involved in APRIL-mediated proliferation in T47D breast cancer cells.

Figure 20. Activation of STAT3 and AKT following sAPRIL treatment of breast cancer cells. T47D breast cancer cells were treated with sAPRIL (Adipogen) at different time points and examined for STAT3 Tyr705 and AKT Ser473 and Thr308 phosphorylation as described in Materials and Methods. Total STAT3 and AKT served as loading controls.

4.1 Discussion

APRIL is a TNF ligand that plays an essential role in the maturation and development of immune cells (Planelles 2008). Although APRIL and its known receptors are recognized to be expressed in lymphoid cells, APRIL has also been detected in certain cancer cells and/or in their surrounding microenvironment (Deshayes et al. 2004, Pelekanou 2008, Planelles et al. 2008, Mhawech-

Fauceglia et al. 2006, Mhawech-Fauceglia et al. 2008,Okano et al. 2005, Wang G. et al. 2013b).

Several studies have demonstrated the involvement of APRIL in biological functions such as proliferation, survival and metastasis of certain cancer cells (Planelles 2008, Quinn et al. 2011).

However, APRIL has also been linked to cell growth arrest (Notas et al. 2012, Wang et al. 2013a) and apoptosis (Roth et al. 2001, Wang et al. 2013a). In breast cancer, APRIL was detected in 50 out of 130 tumor samples (38%) (Mhawech-Fauceglia et al. 2006). APRIL was detected in the tumor stroma but not in the tumor cells themselves except for one sample. Neutrophils in the tumor stroma appeared to be the source of APRIL (Mhawech-Fauceglia et al. 2006). However, the role of APRIL in breast cancer is still unclear. Preliminary data from our laboratory showed that

APRIL was not detectable in the T47D, MCF7 and Hs 578T breast cancer cells. When T47D cells were treated with sAPRIL, increased proliferation of these cells was observed. With 38% of breast tumor cases exhibiting the presence of APRIL in their tumor stromas (Mhawech-Fauceglia et al.

2006), our laboratory’s findings call for further investigation of the role of APRIL in breast cancer.

4.1.1 Impact of APRIL on breast cancer cell proliferation

My initial study was directed at establishing the proliferation effect of APRIL in breast cancer cells. The T47D breast cancer cell model was used to allow continuity of our laboratory’s preliminary investigation. Consistent with our previous data, I found that APRIL enhances

proliferation of breast cancer cells. This was validated using four approaches: (a) transfection of

the cancer cells with APRIL, (b) treatment of the cancer cells with a commercial sAPRIL, (c)

treatment of the cancer cells with sAPRIL that I purified, and (d) loss of proliferation effect of

APRIL inhibited by sAPRIL-BP. The APRIL blocking peptide, sAPRIL-BP, was recently found

to suppress APRIL-induced proliferation in colorectal cancer cells (X-q et al. 2015). My studies

showed a dose-dependent inhibitory effect of sAPRIL-BP on APRIL-induced proliferation in

breast cancer cells. It is interesting that the recently designed sAPRIL-BP (X-q et al. 2015) is

currently proposed as a potential therapeutic agent for colorectal cancer types that shows high level

of APRIL expression and resistance to the drug 5-Fluorouracil (5-FU). Thus, there is a possibility

that down the line, sAPRIL-BP or a similar APRIL blocking peptide could attract attention in the

potential search for therapy of APRIL-mediated breast cancer.

4.1.2 Isolation of sAPRIL

It has been reported that the recognized immune cell receptors for APRIL (TACI and BCMA) have not been identified in breast cancer (Pelekanou et al. 2008). Thus, to investigate the specific function of APRIL in breast cancer cells, one of my goals was to identify and characterize the targets of APRIL in these cells. For this purpose, the use of sAPRIL is required. While I have effectively used commercially available sAPRIL for my studies, the cost of this recombinant protein limits continuous use of the product. I have also effectively used sAPRIL that I have successfully expressed in HEK 293 cells and purified using an antibody based purification system:

FLAG-sAPRIL purification via FLAG antibody attached to agarose. This method, however, had very low yield. Studies in insect cells (Schmidt et al. 2012) suggest that a post-translational modification prevents the FLAG-anti-FLAG interaction making the FLAG tag system ineffective

for secreted proteins such as the FLAG-sAPRIL expressed and secreted by HEK 293 cells.

Apparently, tyrosine in DYK, a critical FLAG epitope, is very susceptible to sulfation that is

catalysed by the tyrosylprotein-sulfo-transferases (Schmidt et al. 2012). It was demonstrated that

this post-translational modification could result in significant loss (less than 20%) of secreted

FLAG- tagged protein being available for binding to anti-FLAG (Schmidt et al. 2012). This modification may explain why my purification of HEK 293 secreted FLAG-sAPRIL using a FLAG antibody yielded very low amounts of protein.

In an effort to obtain sufficient amount of sAPRIL for my studies, I utilized different approaches to purify HEK293-expressed sAPRIL that is secreted into the culture media: (a) DEAE anion exchange chromatography, (b) heparin affinity chromatography, (c) gel filtration chromatography, and (d) ammonium sulfate precipitation. Being secreted into the cell culture media, however, means isolation of sAPRIL from a considerable amount of BSA that originates from the bovine serum that was added to the HEK293 culture media.

It is surprising, however, that BSA, although at high levels, hindered purification of HEK 293- expressed sAPRIL. By DEAE anion exchange chromatography, it seemed that the substantial amount of BSA as well as other proteins in the sample media competed with sAPRIL for DEAE binding. Furthermore, sAPRIL that bound to DEAE mostly coeluted with BSA. Thus, DEAE chromatography yielded a low amount of sAPRIL that was considerably contaminated with BSA.

As with DEAE chromatography, heparin affinity chromatography showed that BSA and other culture media proteins competed with sAPRIL for heparin binding, and lack of sAPRIL in the

NaCl eluates indicated no sAPRIL binding to heparin. Increasing the resin volume or connecting

the DEAE and heparin columns also yielded low amounts of sAPRIL and contamination with BSA

remained significant. Similarly, BSA contamination was evident from sAPRIL isolation by gel

filtration chromatography. Culturing the sAPRIL-expressing HEK 293 cells in low serum media without phenol red, which seemed to also cofractionate with sAPRIL during the purification procedures, still yielded low amounts of sAPRIL with considerable BSA contamination. Together, these chromatography results indicated the use of another approach to purify sAPRIL.

Ammonium sulfate precipitation is widely used in protein purification. In fact, studies have demonstrated that TNFα could be isolated in the range of 40% to 80% ammonium sulfate (Kenig

et al. 2008, Rees et al. 1999). Since APRIL is a member of the TNF superfamily, I sought to purify

sAPRIL by ammonium sulfate precipitation. However, as with the different chromatography

procedures that I performed, BSA cofractionated with sAPRIL. A striking observation was that

the majority of BSA coprecipitated with sAPRIL. My observations raised the idea that sAPRIL

has high affinity to BSA. Potentially, this may be linked to previous studies (Kratz and Elsadek

2012, Shahzad et al. 2014, van der Vusse 2009, and Xu et al. 2012) indicating that BSA is involved

in the cellular transport of biomolecules. Although interesting and possibly relevant, to test these

ideas is beyond the scope of my studies.

To eliminate the problem of BSA contamination, I proceeded to generate bacterially- expressed

GST-sAPRIL. However, as with the purification of HEK 293-expressed FLAG-sAPRIL, the

challenge was to obtain a higher yield of GST-sAPRIL. Since purification of GST-sAPRIL revealed that most of the protein was in the bacterial lysate pellet, this indicated that expressed

GST- sAPRIL accumulated in the insoluble inclusion bodies. Unfortunately, extraction of GST-

sAPRIL from these inclusion bodies using two commonly used concentrations of SDS (0.05% and

0.075%) yielded very low amounts of GST-APRIL. Although 6M to 8M urea could successfully

extract GST-sAPRIL from the inclusion bodies, renatured GST-sAPRIL with potentially altered function would not be useful for my additional studies. Thus, to potentially increase the GST- sAPRIL yield from the bacterial lysate supernatant, bacteria was cultured at a lower temperature

(26°C) and induced to express GST-sAPRIL at a lower concentration of IPTG (50 uM). This method, however, still resulted in a 0.1% yield.

4.1.3 Identification of APRIL-interacting proteins in breast cancer cells

As indicated above, APRIL has been detected in 38% of breast tumors but the protein was found

in the tumor stroma rather than in the tumor cells themselves (Mhawech-Fauceglia et al. 2006).

Although preliminary studies in our laboratory showed that APRIL promotes proliferation of

breast cancer cells, it was reported that the known receptors for APRIL are absent in breast cancer

cells (Pelekanou et al. 2008). Thus, I aimed to identify the targets of APRIL in breast cancer cells using the FLAG-sAPRIL and GST-sAPRIL that I purified as well as a commercially available

FLAG-sAPRIL. A combination of affinity binding to these sAPRIL preparations through precipitation, chromatography or far western blotting and mass spectrometry were performed.

Following affinity binding of T47D breast cancer cell lysate to FLAG-sAPRIL attached to anti-

FLAG-agarose, mass spectrometry identified eight proteins that bound to the affinity column, including CSF2RB, a putative 2.2 kDa protein, serum albumin and mutant beta actin. On the other hand, affinity binding of breast cancer cell lysate to GST-sAPRIL attached to GSH-agarose, identified 30 proteins that bound to the affinity column, including the putative 2.2 kDa protein,

serum albumin and mutant beta actin. Using commercial FLAG-sAPRIL (Adipogen) as bait for far western blotting against breast cancer cell lysate (prey), 150 FLAG-sAPRIL interacting proteins were identified by mass spectrometry, which again included CSF2RB and the putative 2.2 kDa protein. Analysis of the identified APRIL-interacting proteins revealed that these proteins can be classified into 31 groups based on cell function. For example, breast epithelial antigen

BA46, ras-related protein Ral-B, ribosomal protein S15a, and CSF2RB are regulators of cell proliferation; macrophage migration inhibitory factor (Mif) and dermcidin isoform 1 preproprotein are modulators of cell survival and; cytochrome c, mono-ADP-ribosyltransferase, PAP-inositol-

1,4-phosphatase and porin 31HM are regulators of apoptosis. My data indicating that APRIL interacts with proteins that have been implicated in proliferation, survival and apoptosis point to a role of APRIL in these cellular events.

4.1.4 CSF2RB

The identification of CSF2RB, the putative 2.2 kDa putative protein, serum albumin, and mutant beta actin as sAPRIL-interacting proteins in at least two of the three approaches that I used suggests that these proteins are likely to be true APRIL-interacting proteins. However, since I found by multiple sequence alignment and database search that CSF2 (GM-CSF), a CSF2RB ligand, is highly similar to APRIL, I began to characterize APRIL interaction with CSF2RB. Consistent with the affinity binding and far western blotting analyses data, co-immunoprecipitation of APRIL and CSF2RB from HEK 293 cells expressing both of these proteins indicates that they do interact with each other. Further analysis by far western blotting indicates that this interaction is direct.

CSF2RB is already known to be a common beta subunit of the CSF2, IL3 and IL5 cytokine receptors (Broughton et al. 2012, Guthridge et al. 1998, Hercus et al. 2013) but it now appears that

these cytokines also share CSF2RB with APRIL to serve as the APRIL receptor or part thereof in non-immune cells. Further investigation is required to verify this premise. I note, however, that

CSF2RB has 25% similarity to the immune cell APRIL receptors, TACI and BCMA.

Interestingly, residues 38 to 103 and residues 233 to 264 in CSF2RB show partial similarity to the

DXL motif, the binding site of APRIL, in TACI and BCMA, respectively, providing a clue on the potential APRIL binding site in CSF2RB.

4.1.5 PI3K/AKT and STAT3 pathways

The PI3K/ AKT pathway has been implicated in APRIL-induced proliferation of B cells as well as colon cancer cells (Gupta et al. 2009, Wang et al. 2013a, Wang et al. 2013b). In breast cancer, the signaling mechanism for APRIL-mediated proliferation is still unclear but it is recognized that as with other solid and hematological tumors such as ovarian carcinoma, renal carcinoma and multiple myeloma, STAT3 activation induce in breast carcinoma (Banerjee and Resat 2016, Levy and Lee 2002, Gkouveris et al. 2015). Incidentally, both the PI3K/AKT and STAT3 pathways have been linked to cell signaling pathways of CSF2RB (de Groot et al. 1998), which I have identified as an APRIL target in breast cancer cells. CSF2RB has been shown to be involved in several biological functions such as cell proliferation, survival and differentiation through the activation of these pathways (Broughton et al. 2012, Guthridge et al. 1998, de Groot et al. 1998, Hercus et al. 2013). Interestingly, it has been reported that CSF2RB is detectable in various cancer cells, including in certain breast cancers but is absent in normal epithelial breast tissue (Guthridge et al.

1998). Thus, the function of CSF2RB in breast cancer cells is intriguing but examination of this idea is beyond the scope of my investigation.

Consistent with the idea that APRIL acts to induce proliferation of breast cancer cells through a

CSF2RB mechanism, I found that both the AKT and STAT3 pathways are activated in these cells

following treatment with APRIL. As with previous studies (Banerjee and Resat 2016, Levy and

Lee 2002, Gkouveris et al. 2015), I found constitutive but low activation of STAT3 in breast cancer

cells but treatment with APRIL enhanced this activation. A similar observation was found with

AKT activation. Further studies are required to support my theory that APRIL induces

proliferation of breast cancer cells via CSF2RB and the PI3K/AKT and STAT3 pathways.

Nonetheless, my studies revealed CSF2RB as a candidate novel receptor for APRIL in non-

lymphoid cells. Thus, CSF2, IL-3 and IL-5 may share CSF2RB with APRIL to control cell proliferation in breast cancer cells.

4.2 Summary and Future Direction

My studies have established a role for APRIL in breast cancer cell proliferation. This was achieved using the T47D breast cancer cell model that was either transfected or treated with APRIL in

various forms: full-length or soluble, FLAG-tagged or GST-tagged, those that I generated and those obtained as a gift or commercially. My results were supported by my observation that an

APRIL-specific blocking peptide inhibited APRIL-induced proliferation of breast cancer cells. I

have also identified novel targets of APRIL in these cells, including a putative 2.2 kDa protein,

mutant beta actin and CSF2RB. I determined that the CSF2RB ligand, CSF2, has 41% homology

to APRIL, and APRIL interacts with CSF2RB directly, raising the idea that CSF2RB serves as a

receptor for APRIL. Thus, CSF2RB could be a novel APRIL receptor, particularly in non-immune

cells. Consistent with the implied CSF2RB signaling through PI3K/AKT and STAT3 (de Groot

et al. 1998), I found activation of these pathways in APRIL-mediated breast cancer cell

proliferation. Together, my findings have led to new ideas and research directions.

The first logical step is to examine the level of CSF2RB expression in T47D and other breast

cancer cells, and correlate their level of expression with their response in terms of proliferation

upon treatment with sAPRIL. To characterize the involvement of CSF2RB in APRIL-induced

breast cancer cell proliferation, the effect of CSF2RB silencing will be examined. A parallel

analysis of the PI3K/AKT and STAT3 pathways in these cells will also be examined. Since I

found that both the PI3K-AKT and STAT3 pathways are activated in APRIL-treated cells, loss of

activation in APRIL-treated cells depleted of CSF2RB will indicate the requirement for CSF2RB

in the PI3K/AKT and STAT3-mediated APRIL-induced proliferation. Since I determined that

APRIL interacts directly with CSF2RB, it would be interesting to determine the APRIL binding

site in CSF2RB and vice-versa. Homology between CSF2RB and the known receptors for APRIL

in immune cells, TACI and BCMA, will be explored, and potential overlap in CSF2RB and TACI

or BCMA binding sites in APRIL will be examined. Affinity of CSF2RB vs TACI and BCMA to

APRIL will be investigated. A role for CSF2RB in APRIL-induced proliferation of other cancer

types such as colorectal cancer can further be investigated. Since I also identified a putative 2.2

kDa protein, which represents a small part of the T-cell receptor (Koop et al. 1994), as another

APRIL -interacting protein, a potential future research project would be to also characterize the role of this putative protein in APRIL-induced proliferation of breast cancer cells. Studies similar to the ones described above can be performed. This putative protein could possibly be a candidate novel receptor for APRIL in non-lymphoid cells as well.

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