OASIS AND XBP-1 ACTIVITY IN OSTEOBLAST DIFFERENTIATION AND

OSTEOSARCOMA

By

AARON BRADFORD BRISTER

Submitted in partial fulfillment of the requirements

For the degree of Master of Science

Department of Physiology and Biophysics

CASE WESTERN RESERVE UNIVERSITY

January, 2008 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

Aaron Bradford Brister

Candidate for the Master’s degree.

(signed) Corey Smith (Chair of the Committee)

Clark Distelhorst

Edward Greenfield

George Dubyak

Stephen Jones

Susanne Mohr

(Date) 11/20/2007

2 TABLE OF CONTENTS

TABLE OF FIGURES ...... 5

ACKNOWLEDGEMENT ...... 6

Abstract ...... 7

1. Introduction ...... 8

2. Osteoblast Role in Bone Matrix Production ...... 9

3. The Endoplasmic Reticulum ...... 11

3.1. The Unfolded Protein Response ...... 12

3.1.1. Protein Folding and ER Chaperones ...... 15

3.1.2. UPR Stress Sensor Activation and OASIS Relevance ...... 16

3.1.3. UPR Transducers: ATF6, PERK, and IRE1 ...... 18

3.1.4. XBP-1: A UPR Stress Signaling Molecule ...... 19

3.1.5. OASIS: A UPR Stress Transducer ...... 21

4. The Role of OASIS and XBP-1 in Osteoblast Bone Matrix Secretion ..... 24

5. Known Characteristics of Osteosarcoma ...... 29

6. Hyper OASIS and XBP-1 Activity: Enabling Osteosarcoma via Elevated

GRP78? ...... 29

6.1. The Role of GRP78 in Cancer Progression and Metastasis ...... 31

6.2. Mechanisms of GRP78 Cell Death Inhibition ...... 32

6.3 Mechansims of Upregulating GRP78 ...... 33

6.4. The Link between Dedifferentiated Osteoblasts, OASIS, and XBP-1 ...... 34

6.4.1. The Genetic Links between OASIS and GRP78 Overexpression ...... 35

3 6.4.2. The Link between XBP-1 and GRP78 Overexpression ...... 36

7. Conclusions ...... 38

8. Future Directions ...... 40

References ...... 53

4 TABLE OF FIGURES

Figure 1 | Osteoblast differentiation signaling: from mesenchymal stem cells (MSCs)

to mature osteoblasts...... 46

Figure 2 | The pathways within the endoplasmic reticulum (ER) that assist protein

quality control...... 47

Figure 3 | Endoplasmic reticulum (ER) stress and Unfolded Protein Response

signaling...... 48

Figure 4 | Frame switch splicing of XBP-1 by activated IRE1...... 50

Figure 5 | A model of osteoblast differentiation signaling inducing OASIS and XBP-

1...... 51

Figure 6 | A hypothetical mechanism of Retinoblastoma Protein (pRB) inactivation

conferring increases in OASIS and XBP-1 expression...... 52

5 ACKNOWLEDGEMENT

I would like to thank my parents Aubrey and Denise Brister, my brother Matthew, and my girlfriend Lauren Ebling for their love, unfailing support, and encouragement.

6 OASIS and XBP-1 Activity in Osteoblast Differentiation and Osteosarcoma

Abstract

By

AARON BRADFORD BRISTER

The Unfolded Protein Response (UPR) within the Endoplasmic Reticulum (ER) is a quality control mechanism ensuring properly folded proteins. OASIS and XBP-1 are two signal carriers of the UPR. The UPR is linked to tissue and cancer development.

Elevated OASIS and XBP-1 activity are observed within developing osteoblasts.

Additionally, heightened OASIS activity is present within osteosarcoma while sustained

XBP-1 activity, unreported in osteosarcoma, is observed in other cancers. These signaling proteins may mediate development of mature osteoblasts and osteosarcomas.

Therefore, evidence is presented with supporting mechanistic hypotheses indicating unique functions for OASIS and XBP-1 in osteoblast differentiation and osteosarcoma.

These functions include, but are not limited to, enlarging the ER, buffering unfolded protein accumulation, mitigating UPR-induced cell death, and processing soluble ER proteins destined for secretion.

7 1. Introduction

The intent of this document is to examine two signaling molecules associated with the

Unfolded Protein Response (UPR), a quality control mechanism within a cell’s

Endoplasmic Reticulum (ER). These are Old Astrocyte Specifically Induced Substance

(OASIS) and X-box Binding Protein 1 (XBP-1). OASIS and XBP-1 activities will be

discussed within two contexts: osteoblast differentiation and osteosarcoma. Each context

will address one of the following questions. Are OASIS and XBP-1 activities directed by

osteoblast differentiation programs and do their activities promote protein folding for

osteoblast secretion? Could OASIS and XBP-1 hyperactivity enable osteosarcoma?

The first question concerning osteoblast differentiation will be addressed in sections two

through four and develop the following novel hypotheses. First, in differentiating

osteoblasts, a sublethal UPR initiated by progressive increases of protein production

upregulate OASIS and XBP-1 expression levels. Secondly, in maturing osteoblasts, the

signaling pathways driving osteoblast differentiation also induce OASIS and XBP-1

expression so progressive increases of protein expression are matched with increases of

OASIS and XBP-1 expression. Alternatively, both hypotheses may occur together.

Regardless, either hypothesis prepares an osteoblast’s ER for elevated protein production

and subsequent secretion, their primary functions. Further hypotheses will suggest

specific functions of OASIS and XBP-1 in assisting protein processing through the ER.

The second question concerning osteosarcoma will be addressed in sections five through

six and develop the following hypotheses. First, in osteosarcoma, hyper OASIS and

8 XBP-1 activity maintains steady-state protein folding within the ER so an UPR once sufficient to induce cell death is now sublethal. Secondly, if differentiating osteoblasts do upregulate OASIS and XBP-1, then perhaps osteosarcomas may exploit this pathway to maintain steady-state protein folding within the ER and mitigate cell death. Additional hypotheses will examine how OASIS and XBP-1 hyperactivity reduces an UPR to a sublethal level. Also, an additional hypothetical mechanism of how OASIS is overexpressed in osteosarcoma will also be proposed.

2. Osteoblast Role in Bone Matrix Production

Bone is composed of cell types derived from an embryonic germ layer. The mesoderm is an embryonic germ layer which produces Mesenchymal Stem Cells (MSCs). MSCs are self-renewing and multipotent in nature. Otherwise stated, MSCs regenerate through mitotic division or give rise to many cell types. MSCs develop into osteoblasts, bone forming cells that synthesize and secrete osteoid tissue (see figure 1). Osteoid tissue is created when proteins exuded by osteoblasts, together with minerals, form bone scaffolding. Bone proteins interact with calcium, magnesium, and phosphate ions forming hardened mineral matrix, i.e. hydroxyapatite. After an osteoblast finishes forming bone it becomes quiescent, also known as a lining cell [1, 2].

MSCs become mature osteoblasts which are “fully differentiated cells responsible for the production of the bone matrix” [3]. MSCs become osteoblasts through a complex differentiation program and many markers and signaling pathways of this program have been characterized. These markers are available for analysis and indicate the progression

9 of MSC differentiation. Once a MSC becomes a differentiating osteoblast it actively

synthesizes and simultaneously secretes osseous tissue or osteoid matrix proteins which construct the skeletal system. Osseous tissue proteins include, but are not limited to, collagen type 1 IA, Alkaline Phosphatase, Osteopontin, and Osteocalcin. Important signaling molecules within differentiating osteoblasts are Runt-Related Transcription

Factor 2 (RUNX2), Retinoblastoma protein (pRB), and Osterix ([4, 5] and references therein).

A MSC is committed to become an osteoblast when it expresses both the intracellular signaling proteins RUNX2 and Osterix. RUNX2 (or Cbfa1) is a member of the RUNT transcription factor family. RUNT proteins have a DNA-binding domain homologous to the Drosophila pair-rule gene, Runt ([4] and references therein). RUNX2 is a transcription factor essential for osteoblast development. This is based on animal models engineered to lack RUNX2 expression and fail to develop osteoblasts along with their corresponding mineralized bone matrix. For this reason, RUNX2 is named the “master osteoblast regulator” [6-9]. Osterix is a zinc-finger transcription factor promoting osteogenic-commitment, proliferation, and expression of osteoid matrix proteins. When an osteoblast exits the cell cycle it commits to terminal differentiation. The pRB is an important signaling molecule for the cell cycle, survival, and osteoblast differentiation

([4] and references therein). The pRB is a pocket protein shown to physically bind with

RUNX2 in osteoblasts when it is hypophosphorylated [10]. This binding is thought to signal the final stages of osteoblast differentiation (see figure 1). These signaling events are important because they may drive OASIS and XBP-1 expression.

10 Varied extracellular factors contribute to osteogenesis through increasing RUNX2

expression. These factors include glucocorticoids, vitamin D, Ascorbic Acid (vitamin C),

Bone Morphogenetic Proteins (BMPs), Parathyroid Hormone (PTH), and Notch

Signaling (see figure 1). Prominent osteogenic signal carriers include Wnt, TGF-β,

Notch, and MAP-kinase pathways. These factors will upregulate RUNX2. Then

RUNX2 induces expression of Osterix. Expression of both RUNX2 and Osterix signals osteogenic-commitment. The physical interaction of pRB with RUNX2 is thought to signal terminal osteoblast differentiation ([4, 5] and references therein). Progressive

RUNX2, Osterix, and pRB signaling in maturing osteoblasts is linked to increased expression of osseous tissue and a widely accepted model of osteoblast differentiation.

However, this model lacks an explanation of how developing osteoblasts maintain pressures within their ERs from progressive increases of protein expression. This weakness begs the question, how does an osteoblast produce large amounts of osteoid matrix proteins while not perturbing steady-state protein folding within its ER? The consequences of ER stresses are severe and, if not mitigated, could lead to cell death.

Maintaining steady-state protein folding, making sure unfolded proteins do not accumulate within the ER, prevents cell death and points to an important role for OASIS and XBP-1.

3. The Endoplasmic Reticulum

The Endoplasmic Reticulum (ER) is a cellular organelle in eukaryotes that is an interconnected network of vesicles, tubules, and cisternae. The ER serves several specialized functions including the site of protein synthesis, protein folding, drug

11 detoxification, calcium buffering, and others (see figure 2). Extracellular proteins, like osseous tissue, are processed through the ER. The ER requires tight regulation because of its many functions. Protein folding, which is an ER function, is a complex phenomenon requiring tight regulation of its parameters. These folding parameters direct hydrophobic interactions, hydrogen bond formation, and van der Waals forces required for specific protein folds. Opposing these forces is the conformational entropy of an unfolded protein. Various ER-resident proteins are charged with assisting protein folding. The chemistries within the ER are also important to ER-resident proteins.

Solutes in the ER, together with ER-resident proteins, assist folding proteins. For instance, ER-resident enzymes may modify a protein with a sugar side group to direct its folding. On the other hand, a protein may be modified through an enzymatic-redox reaction producing a disulfide bond on a folding protein. As such, the ER requires specific parameters. If these parameters become deregulated, then the ER is stressed and cannot fold proteins correctly, causing unfolded and/or misfolded proteins to accumulate within the ER. One mechanism evolved and employed within the eukaryotic ER to counter act these stresses is the UPR ([11] and references therein).

3.1. The Unfolded Protein Response

The UPR is a reaction to changes in the steady-state levels of protein folding produced by a stressor. In other words, a shift in equilibrium within the ER that favors unfolded versus folded proteins (see figure 3). Eukaryotic cells adapted the UPR to counter act a stressor that favors unfolded protein accumulation. ER stressors that produce an UPR include an increased rate of newly synthesized protein entering the ER (nascent protein),

12 saturation of folding chaperones, irregular solute levels, and an accumulation of unfolded

proteins. If these parameters are not maintained, then the mechanisms directing proteins

to their correct conformation work improperly. Therefore proteins will misfold, remain

unfolded, or become unfolded. This event is perceived by stress-sensors of the ER and

signaled as an UPR. The cell counters unfolded protein accumulation with three

immediate UPR interventions: decreasing the rate of proteins entering the ER, increasing

protein folding rates, and increasing degradation of unfolded proteins. Other UPR

intervention targets include nuclear transcription of specific ER-resident proteins,

autophagosome formation, and expansion of the ER. However, if a cell encounters ER-

stress that is excessive or long-lived, then the result is programmed cell death or

apoptosis ([12-18] see figure 3).

The UPR has a role in growth and development [19], enhancing ER volume [20],

increasing protein secretion, and scaling-up the amount of proteins processed through the

ER [21]. UPR signaling influences transcription [22-24], translation [24], apoptosis [17],

and autophagy [25]. Autophagy is a mechanism whereby a cell creates autophagasomes

that digest cellular proteins during conditions of low nutrient availability. Other ER stressors include ER Ca2+ depletion, inhibition of N-linked glycosylation, reduction of

disulfide bond formation, protein overexpression, and solute depletion. Solute depletion

includes changes in glucose, ATP, or oxygen levels ([12-18] see figure 2). Each stressor

can tailor an UPR. A tailored response is based on intrinsic properties of the ER-stress

transducers [18]. These transducers or stress-sensors are transmembrane proteins

localized to the ER that signal an UPR.

13 The UPR has roles in health and disease. For instance, the UPR may be linked with

cancer through its signal carriers [16, 26, 27]. Furthermore, the UPR is implicated in the

pathogenesis of neuronal degenerative diseases such as Alzheimer's [28] and Parkinson's

disease [29]. The UPR is also linked with diabetes [30], viral pathogenesis [31], and

some genetic diseases [13] such as Huntington’s Disease [32], and a bone disease known as Wolcott-Rallison Syndrome [33]. A number of osteoblast differentiation and UPR studies are in the literature; however, no study has linked osteoblast differentiation with osteosarcoma through the UPR despite evidence of its activation [34-39].

One study did examine osteoblast maturation and UPR signaling. In osteoblasts, if ER- stress is induced by calcium depletion or inhibition of N-linked glycosylation, then a biphasic response is observed. This response is first marked by activated eukaryotic

Initiation Factor 2α (eIF2α) as well as induction of Activating Transcription Factor 4

(ATF4), Osterix, and RUNX2. Both eIF2α and ATF4 have a role in UPR signaling. If these ER-stressors are maintained for long periods (about 24 hours), then apoptosis occurs [40]. Because ER-stress induces expression of Osterix and RUNX2, which are two signaling molecules that direct osteoblast differentiation, it is evident the UPR communicates with osteoblast differention pathways. The question arises, what is the function of this signaling? Perhaps this signaling is a positive feedback mechanism reinforcing osteogenic-commitment. Could communication between the UPR and osteoblast maturation promoters further maintain the steady-state levels of protein folding within the ER? Since osteoblasts primarily secrete extracellular bone proteins and if

RUNX2 and Osterix signaling drive OASIS and XBP-1 expression, then perhaps this

14 cross signaling promotes osteogenic-commitment and maintains steady-state protein

folding in the ER.

3.1.1. Protein Folding and ER Chaperones

Just as RUNX2 is the master osteoblast regulator, Glucose Regulatory Protein 78

(GRP78), or Binding Protein (BiP), is the “central regulator of the ER” [15]. Another

important ER regulator is Glucose Regulatory Protein 94 (GRP94). Both GRP78 and

GRP94 are soluble proteins localized to the ER-lumen. These proteins are chaperones

and catalytic enzymes that assist in calcium sequestration, protein folding, and quality

control (see figure 2). A widely accepted mechanism for activating the UPR requires

GRP78. GRP78, when bound to the ER-stress transducers prevents their activation. If

GRP78 dissociates from these ER-stress transducers, then they become active [15, 18,

41].

The importance of GRP78 and GRP94 to ER homeostasis is underscored by the presence

of ER Stress Response Elements (ERSE) and UPR Elements (UPRE) within the promoter

regions of their genes [22]. These genetic elements are targeted by UPR signaling. Thus,

GRP78 and GRP94 proteins are recruited immediately following an UPR to assist protein

folding.

Induction of GRP78 and GRP94 occurs despite cellular reductions to overall protein synthesis. However, a more immediate recruitment of GRP78 comes from the pool of

GRP78 bound to ER-stress transducers. This GRP78 pool buffers an unfolded protein

15 accumulation. In other words, if there is ER-stress, then GRP78 dissociates from the ER- stress transducers to mitigate unfolded protein accumulation. Once GRP78 dissociates from these ER-stress sensors an UPR can be signaled. This signaling occurs because ER- stress sensor activity is no longer inhibited by GRP78 [15, 18, 41].

3.1.2. UPR Stress Sensor Activation and OASIS Relevance

The current model of ER-stress communication to the ER-stress transducers involves

GRP78. This model is supported by the observation that GRP78 binds with, and regulates the activity of, the ER-stress transducers in non-stressed cells [13, 42, 43]. This suggests a competitive binding mechanism of UPR activation. When unfolded or misfolded proteins accumulate within the ER they out-compete the ER-stress transducers for GRP78 binding. Since GRP78 is no longer physically bound to these stress sensors they become active (see figure 2). This model is consistent with observations of Protein

Kinase R-like ER-localized eIF2α Kinase (PERK) and Inositol requiring protein 1 (IRE1) activation. Both PERK and IRE1 are ER-stress transducers of the UPR. When GRP78 is bound to these stress-transducers it sterically blocks the formation of their homodimers.

Thus, bound GRP78 prevents PERK and IRE1 activation (see figure 3). Evidence supporting this mechanism has been demonstrated by deletion of the PERK N-terminus or its ER luminal domain. Since this PERK construct is unavailable for GRP78 binding in the lumen, PERK is constitutively activated [44].

This widely accepted mechanism, however, is increasingly disputed. There are reports of tailored UPRs to specific stressors. Evidence of ER-stress transducer activation

16

(following ER calcium perturbations, oxidation, or inhibition of peptide glycosylation) established that each has distinct sensitivities for different forms of stress. The proposed mechanism involves structural changes within the ER-stress transducer adjusting its

GRP78 binding affinity ([18] see figure 3).

Another mechanism of UPR activation, which has implications for OASIS, comes from the following. Studying Activating Transcription Factor 6 (ATF6) activation, an ER- stress transducer, illustrates a unique GRP78 regulatory model (see figure 3). ATF6 does not homodimerize to become active. Further, the ATF6-GRP78 complex was easily isolated without the use of cross-linking agents. This is consistent with GRP78 regulation of ATF6 activation, but suggests very stable binding because many protein complexes require chemical cross-linking prior to isolation. ATF6 binds wild-type

GRP78 at similar levels to mutant GRP78 lacking ATPase activity. This activity enhances GRP78 binding to ATF6 which is not reversed by the addition of ATP. An

ATF6 luminal domain is required for ER-stress-triggered GRP78 release. From these data, it was suggested GRP78 actively dissociates from ATF6. In other words, ATF6 restarts GRP78’s ATPase cycle causing its dissociation [45]. Perhaps OASIS acts similarly because ATF6 and OASIS share considerable homology, structure, regulation, and function [46]. Therefore, the interaction of ATF6 regulating GRP78 binding, by analogy, may occur between OASIS and GRP78. If correct, this analogy may have an important role in protein folding within an osteoblast’s ER.

17 3.1.3. UPR Transducers: ATF6, PERK, and IRE1

The functions of ATF6, PERK, and IRE1 are outlined here. ATF6 is a membrane-bound transcription factor (see figure 3). ATF6 is a type II transmembrane protein localized to the ER. ATF6 contains a basic leucine zipper (bZIP) transcription factor. When there is

ER-stress, ATF6 translocates to the Golgi apparatus and is processed by regulated intramembrane proteolysis (RIP). The Golgi contains Site-1 and Site-2 proteases (the

RIP mechanism) which cleave the cytosolic ATF6’s bZIP transcription factor from the rest of the protein [47, 48]. Cleaved ATF6, liberated from the cytosolic surface of the

ER, translocates to the nucleus and is designated pATF6 (N). When in the nucleus, the bZIP transcription factor of pATF6 (N) can bind genomic elements (such as ERSEs and

UPREs) and induce GRP78 and XBP-1 expression [22, 49].

The molecule PERK is a type I transmembrane ER-stress transducer of the UPR localized to the ER (see figure 3). PERK signaling evokes a transient inhibition of protein synthesis which decreases protein influxes into the ER. When PERK senses ER-stress, two PERK monomers dimerize and autophosphorylate one another. Phosphorylation occurs on their cytosolic kinase domains activating PERK [50, 51]. When active, PERK phosphorylates the alpha subunit of Eukaryotic Initiation Factor 2 alpha (eIF2α).

Phosphorylation of eIF2α, a soluble protein localized to the cytosol, activates it.

Phosphorylated eIF2α inhibits protein translation by physically blocking the formation of the ribosome’s 43S translation-initiation complex, thus inhibiting protein synthesis [24].

Paradoxically, PERK activity also induces protein synthesis. PERK-mediated protein synthesis produces a phosphatase that dephosphorylates eIF2α. Otherwise stated, it

18 creates a negative feedback loop [52]. Following ER-stress, PERK activity inhibits

cellular proliferation by causing G1 cell cycle arrest [53] and induces CHOP (C/EBP-

homologous protein) expression [54], a protein implicated in apoptosis [55]. These

observations are important to osteoblast biology because inactivation of the cell cycle is

required for terminal differentiation. Also, because PERK activity mediates both

upregulation of RUNX2 and Osterix signaling and, if persistent, apoptosis [40].

The IRE1 molecule is a type I transmembrane protein of the ER with kinase and endoribonuclease domains on its cytoplasmic C-terminal ([56, 57] see figure 3). IRE1 becomes active when two IRE1 monomers dimerize and autophosphorylate [50, 51].

This protein has a unique feature because of its endoribonuclease (RNase) activity which, after activation, mediates frame switch splicing of XBP-1 [58]. Frame switch splicing will be further discussed below.

3.1.4. XBP-1: A UPR Stress Signaling Molecule

All adult tissues ubiquitously express XBP-1 [20]. Despite XBP-1’s innocuous expression pattern, its regulation is unique (see figure 4). In eukaryotes, most primary transcripts (pre-messenger or pre-mRNA) contain introns, which are subjected to spliceosome-dependent splicing reactions or alternative splicing. Alternative splicing is a

nuclear-event. This splicing is followed by the transport of mature or spliced mRNA

(intron-free) into the cytoplasm. After mature mRNA enters the cytoplasmic space its

directionality is utilized by a ribosome to translate mRNA into protein [59].

19 An exception to this model exists in XBP-1 regulation. XBP-1 mRNA is a substrate for

both IRE1 and a ribosome. XBP-1 mRNA is unusual because of the unconventional

mRNA splicing that centers around its activity state. XBP-1 pre-mRNA is subject to

alternative splicing. After XBP-1 mRNA localizes to the cytoplasm it can either be

translated by the ribosome or targeted by active IRE1. XBP-1 mRNA codes for two

different C-terminal peptide sequences starting at a 26 nucleotide intron-like sequence.

One of these distinct C-terminal XBP-1 peptide sequences is expressed under basal

conditions, while the other is expressed following ER-stress. ER-stress causes the

expression of a distinct XBP-1 C-terminus after active IRE1 targets the 26 nucleotide

sequence [23, 60]. This 26 nucleotide region of XBP-1 mRNA contains a characteristic stem-loop structure, similar to HAC1 mRNA [61, 62]; a functional orthologue of XBP-1 found in budding yeast [63, 64]. This XBP-1 stem-loop structure is targeted by active

IRE1 as its substrate.

After ER-stress activates IRE1, this protein targets the 26 nucleotide intron-like sequence in the middle of XBP-1 mRNA. Then, two pieces are digested away from the middle 26 nucleotide sequence. These flanking pieces are ligated together by a tRNA ligase, Rlg1p

[65]. This cleavage and subsequent ligation step produces a XBP-1 mRNA sequence, minus the 26 nucleotide intron-like sequence. Removing these 26 nucleotides causes a reading frame shift. In other words, digestion and subsequent ligation of XBP-1 mRNA shifts its codons after the 26 nuceleotide sequence. An mRNA reading frame shift codes for a different XBP-1 C-terminal peptide sequence when it is expressed. After a reading frame shift, XBP-1 mRNA codes for a highly active bZIP domain within the C-terminus

20 of this protein. This highly active bZIP domain is only expressed following ER stress

[23]. This novel mechanism of regulating protein activity has been designated ‘frame

switch splicing’ ([60] see figure 4).

The XBP-1 mRNA transcript consists of 1846 nucleotides. Under conditions of no ER

stress the unspliced (U) XBP-1 transcript is translated into a 261 amino acid protein of no

known function. During an UPR, frame switch splicing creates a spliced (S) XBP-1

mRNA transcript. Spliced XBP-1 is translated into a 376 amino acid protein containing a

highly active bZIP transcription factor [23]. Regardless of the C-terminal peptide

sequence of either the spliced or the unspliced XBP-1, the N-terminal region remains

unchanged. When an UPR mediates frame switch splicing of XBP-1, (S) XBP-1 is synthesized and localizes to the nucleus. Then the bZIP transcription factor of XBP-1 binds with UPRE and ERSE of the cells genome inducing GRP78 expression ([22, 66] see figure 3).

3.1.5. OASIS: A UPR Stress Transducer

OASIS exhibits both spatial and temporal expression patterns within mouse tissues. This spatial distribution includes lung, kidney, brain, spleen, liver, thymus, salivary gland, tooth germ, and bone tissues [34, 35, 67]. OASIS was first identified in primary mouse- brain cultures [67]. These cultures were aged several weeks as a model of astrocyte gliosis. Gliosis is a scarring process in the Central Nervous System. Gliosis is

characterized by phenotypic modification of astrocytes, increased cellular proliferation,

and the recruitment of additional cell types to the wounded area. Gliosis is associated

21 with upregulation of several proteins, such as glial fibrillary acidic protein (GFAP).

From this gliosis model OASIS was cloned and given the name Old Astrocyte

Specifically-Induced Substance [67]. OASIS mRNA levels closely correlate with GFAP

levels in injured mouse brains [67]. It is thought that OASIS functions as a transcriptional regulator of gliosis [68]. Glial scarring is also associated with production of chondroitin sulfate proteoglycan, an extracellular protein [69]. Proteoglycans are highly glycoslyated proteins of the extracellular matrix and assembled in the ER.

Additionally, proteoglycans are widely believed to play a role in the calcification of mineralized tissues, such as bone [70]. The hypothesis stated in section 3.1.2 regarding

OASIS interacting with GRP78 through its ATPase cycle, together with the link between

OASIS and proteoglycan secretion, may suggest a specialized function. Perhaps OASIS

and GRP78 interactions are required for producing properly folded proteoglycans for

osteoblast secretion.

Human OASIS consists of a 2701 nucleotide mRNA transcript, which codes for a 519

amino acid protein. At the nucleotide level, OASIS is 63% identical to the Drosophila

Box B Binding factor-2 (BBF-2) gene, a CREB/ATF transcription factor family member

[71, 72]. The cAMP Reponse Element Binding (CREB) family consists of proteins that

bind cAMP Response Elements (CRE) on DNA. At the amino acid level, OASIS is

predicted to be a type II transmembrane protein and 31% identical to ATF6. Because

OASIS shares similar features with ATF6 and BBF-2 it is considered a member of the

CREB/ATF family. Thus, OASIS is also called CREB3L1. OASIS and ATF6 are similar stress sensors [46]. Indeed, OASIS signals unfolded protein accumulation within

22 the ER by cleavage activation, like ATF6. OASIS cleavage can produce one of two

products and either cleavage product comes from the N-terminus of OASIS by a RIP mechanism. However, both cleavage products contain a bZIP transcription factor that

targets UPREs and ERSEs of the genome ([46] see figure 3).

Several transcription factors localize to membranes within a cell, as do ATF6 and

OASIS, and their activity is regulated by a RIP mechanism. The RIP mechanism allows

membrane bound transcription factors to become active after cleavage. RIP cleaves the

transcription factor from the rest of the protein so only the soluble (cytosolic-

proteolyized) fragment can enter the nucleus and subsequently regulate gene transcription

([48] see figure 3). OASIS contains an Arg-Ser-Leu-Leu (RSLL) sequence, beginning at amino acid 423. This sequence fits the RXXL consensus sequence for S1P, a membrane- anchored serine protease whose active site faces the Golgi lumen [46, 68]. S1P is an enzyme involved in RIP and targets the RXXL motif found on both ATF6 and OASIS.

If the RXXL domain of ATF6 is mutated, then ATF6 cleavage is blocked [47]. This demonstrates that S1P proteolysis of ATF6 determines whether it produces a cleavage product. This is in contrast to OASIS. Proteolysis of OASIS occurs once; however,

OASIS can be proteolyzed by either S1P or S2P. Researcher Imaizumi and his colleagues [73] demonstrated that two distinct OASIS proteolytic fragments (one of 55

kilodaltons or another of 50 kilodaltons) are produced. Either S1P proteolysis produces a

55 kilodalton fragment, or S2P proteolysis produces a 50 kilodalton fragment. These

fragments are produced differentially with respect to time following ER stress. To test

23 this differential OASIS cleavage mechanism they targeted the S1P and S2P recognition

sites. By scrambling the RXXL domain, through introduced mutations, inhibits cleavage

of the 55 kilodalton fragment, but produces a 50 kilodalton proteolytic fragment. S1P

can be inhibited pharmacologically with AEBSF. Treatment of AEBSF, prior to ER-

stress, produces only the 50 kilodalton fragment. Next, these researchers targeted the

S2P site. The S2P cleavage site is less defined motif than S1P, yet mutations within this

site inhibit the reaction. Mutating the S2P site on OASIS inhibits S2P-mediated

proteolysis. However, the 50 kilodalton OASIS fragment is produced [73]. Therefore,

OASIS is cleaved by S1P or S2P producing either a 55 or 50 kilodalton fragment (see figure 3). The implications of S1P and S2P cleavage will be further discussed in the future directions section.

Following cleavage of OASIS, both cleavage-fragments (p50 OASIS and p55 OASIS)

translocate to the nucleus and interact with CREs and ERSEs of certain genes. One of

the targets of these cleaved OASIS fragments is the GRP78 gene [46, 73]. The two

OASIS cleavage fragments induce GRP78 expression at different levels [73].

4. The Role of OASIS and XBP-1 in Osteoblast Bone Matrix Secretion

The primary role of osteoblasts is the production of osteoid bone matrix (see figure 5). If

so—then why do the osteoblast differentiation signaling pathways outlined above lack a

mechanism to balance ER homeostasis with the secretory demands of an osteoblast? In

fact, three points suggest such a role and implicate an intimate relationship for OASIS

and XBP-1 in this process.

24 First, it was noted developing mouse embryos concurrently express OASIS and XBP-1 within their osteoblasts [34]. This observation is consistent with in situ hybridization data identifying the bZIP motif of XBP-1’s role in exocrine gland and skeletal development [35]. Exocrine glands produce proteins for secretion. Then, researchers

Toshio Nikaido and colleagues [34] sought to determine test whether the spatiotemporal expression patterns of OASIS and XBP-1 correlated with osteogenesis differentiation markers. This study found that OASIS expresses in rib pre-osteoblasts of the outer bony cortex, alveolar bone, and in preodontoblasts. These researchers noted concurrent OASIS and XBP-1 expression in osteoblasts. From this data these researchers advanced the hypothesis that OASIS and XBP-1, acting cooperatively through their bZIP domains, form homo/heterodimers. This theory is based on data from other bZIP containing proteins (CREB/ATF, Fos, and Jun) which are known to form homo/heterodimers. This hypothesis highlights the importance placed by these researchers on OASIS and XBP-1 concurrent expression; however, this group points-out OASIS and XBP-1 expression patterns are not concurrent in exocrine glands. In osteoblasts, OASIS and XBP-1 concurrent expression closely correlates with osteopontin and osteonectin, which are osteoid matrix protein. Most importantly, this group observed two things during osteoblast maturation. Foremost, RUNX2 (Cbfa1) expression occurs prior to OASIS and

XBP-1 expression. Plus, OASIS and XBP-1 expression occurs before procollagen I type

1α mRNA, the precursor to collagen I type 1α which is the predominant osteoid matrix protein ([34] and references therein). Furthermore, in MC3T3 osteoblast models

Parathyroid hormone (PTH), which is a known osteoblast promoter, increases the levels of XBP-1 [74]. Together, these data are consistent with the following novel hypothesis.

25 Osteoblast differentiation signaling induces OASIS and XBP-1 expression—prior to high levels of osseous tissue expression—because these proteins are responsible for developing high protein folding through-put within an osteoblast’s ER (see figure 5).

Second, during B-cell differentiation XBP-1 mediates a five-fold expansion of their ER

[20]. B-cells are lymphocytes that participate in the humoral-immune response, which requires robust ER development for antibody synthesis. Antibodies are soluble extracellular proteins secreted from B-lymphocytes that assist in the destruction of microbe-pathogens. XBP-1 is also essential to C. elegans development [19] as well as to both plasma cell and hepatocyte differentiation [75, 76]. XBP-1 is thought to scale-up the amount of protein processed through ER [21, 53, 77, 78]. This is illustrated in the

XBP-1 knock-out mouse. This mouse is embryonic lethal. This lethality is linked to hepatocytes [79]. Hepatocytes are highly secretory cells of the liver that produce soluble serum proteins like albumin, fibrinogen, and prothrombin. Hepatocytes also secrete lipoproteins, ceruloplasmin, transferrin, and glycoproteins. Thus, XBP-1 inhibition implies an essential role in hepatocyte secretion. Perhaps all secretory cell types require

XBP-1 activity.

Third, Murakami and colleagues ([36] unpublished data) recently stated they developed an OASIS knock-out mouse. This knock-out mouse has a bone phenotype of growth retardation and abnormal osteogenesis. This group observed that OASIS knock-out mice display both deformity of limb bones and joint swelling. OASIS deficient mice also have less bone mass. Observing their tissue with an electron microscope demonstrated “an

26 accumulation of secreted material within the ER and an expanded rough ER.” The rough

ER is a contiguous membrane of the ER. However, the rough ER is studded with bound ribosomes actively synthesizing protein for the ER processing. These observations underscore an essential OASIS function in the secretion of extracellular proteins from the

ER of osteoblasts. These data suggest one of two things: either protein accumulation in the ER of OASIS-null mice is insufficient to induce cell death or some compensatory mechanism permits osteoblast survival.

All together, these data support a role for OASIS and XBP-1 in osteoblast differentiation.

These data also support three mechanistic hypotheses of how the ER of pre-osteoblasts progressively scale-up to handle the secretory demands of mature osteoblasts. First, the

ER may accommodate increased protein load in the ER of osteoblasts secondary to elevated protein synthesis. If an ER protein influx signals an UPR insufficient to cause cell death, then lower level UPR signaling could upregulate folding chaperones in an osteoblast’s ER. Second, during osteoblast differentiation the signaling pathways that transcriptionally induce bone proteins may, in parallel, induce OASIS and XBP-1 expression which in turn increases the protein folding through-put of the ER (see figure

5). This hypothesis is consistent with both OASIS and XBP-1 concurrent expression with osteopontin in osteoblasts. This hypothesis is also consistent with increased

RUNX2 activity occurring before increased OASIS and XBP-1 expression. This theory is also consistent with OASIS and XBP-1 expression preceding procollagen I type 1α mRNA, the precursor to the primary constituent of osseous tissue [34]. The A third possibility is the first and second hypotheses occur together. This is supported by the

27 biphasic response observed in ER-stress challenged osteoblasts. The initial response to

sublethal ER-stress was the induction of RUNX2 and Osterix proteins [40]. Perhaps ER

stress upregulates RUNX2 and Osterix expression further enhancing osteogenic-

commitment of pre-osteoblasts. If osteoblast differentiation signaling does upregulate

OASIS and XBP-1, then increasing RUNX2 and Osterix provide further positive

feedback.

Clearly, ER-stress communicates with osteoblast differentiation signaling given the

biphasic response study of stress-challenged osteoblasts. If ER-stress continues

unabated, then osteoblasts die by apoptosis [40]. This highlights the importance of

maintaining stresses within the ER of osteoblasts. However, several questions regarding

what OASIS and XBP-1 activity does still remain. What purpose do these signaling

proteins serve osteoblasts? Regarding XBP-1, its activity is linked to hepatocyte

differentiation, the 5-fold expansion of the ER in B-lymphocytes, and the induction of ER

chaperone proteins. These are critical functions to secretory cell types. Thus, it is my

hypothesis that XBP-1 activity elevates the scale at which the ER can fold proteins in maturing osteoblasts by increasing ER’s volume and GRP78 levels. Regarding OASIS, if this protein actively regulates GRP78 dissociation by restarting its ATPase cycle similar to ATF6, then what does this imply? It is known that OASIS knock-out mice accumulate soluble proteins within their ER lumen. Also, OASIS activity both induces GRP78 expression and is linked with glial scar formation. Glial scars are marked by proteoglycan secretion. Therefore, it is my hypothesis that OASIS, through active

28 regulation of GRP78, specializes in processing proteoglycans through the ER. Perhaps the accumulated materials within the ER of OASIS knock-out mice are proteoglycans.

5. Known Characteristics of Osteosarcoma

Many factors are thought to contribute to osteosarcoma. At the genomic level, these factors include mutations, amplifications, and epigenetic silencing. The most frequently affected osteosarcoma targets are the tumor suppressor genes RB1 and TP53. In fact, alterations to the RB1 gene, which cause gene silencing or inactivation, occur in about

90% of primary human osteosarcoma tumors [4]. The RB1 gene codes for the retinoblastoma protein (pRB), the most frequently inactivated cell-cycle regulatory pathway of all human tumors. The cell cycle is a series of events controlling cellular replication and differentiation. This is important because the majority of osteosarcoma tumors are highly undifferentiated [80, 81]. The observations that pRB in healthy bone physically interacts with RUNX2 [10], together with the observation pRB signaling is largely inactive in osteosarcoma [4], has important implications for hyper OASIS and

XBP-1 activity in osteosarcoma.

6. Hyper OASIS and XBP-1 Activity: Enabling Osteosarcoma via Elevated

GRP78?

In a landmark study, a research team lead by Amy Lee and colleagues [26] demonstrated, in a fibrosarcoma cancer cell line, that when GRP78 expression is reduced, or knocked- down, these cells produce fewer tumors when injected into nude mice compared to control-level expressers. The implication of GRP78 inhibition, in Lee’s words, “has a

29 major effect on tumor growth.” Indeed, many cancers and their models (cell lines, solid

tumors, and cancer biopsies) present with highly-elevated GRP78 expression levels.

Elevated GRP78 levels also correlate with malignancy, metastasis, and drug resistance

[82].

Several cellular defenses within the host cell prevent cancer development; a cancer must

counter these to survive. Two important cellular defenses a cancerous cell must control

are the cell-cycle and apoptotic signaling. Mutations within a cancerous cell disabling

the host defenses are extremely beneficial. For instance, inactivation of pRB signaling

deregulates the cell cycle in osteosarcoma, just as controlling the extent of UPR signal

controls apoptosis.

Cancers face other challenges, like nutrient restriction. Nutrient restrictions can be initiated by the host body encapsulating a tumor or from a highly aggressive tumor

outgrowing its blood supply. Thus, nutrient availability can become scarce. This

challenges cancerous cells by causing stress within the ER. If stresses cause unfolded

proteins to accumulate within the ER, then an UPR will ensue. If this ER-stress is

excessive or long-lived, then the cell may die from apoptosis [15-17]. For a cancerous

cell to survive ER-stress a new mechanism of maintaining homeostasis must be

introduced so that an UPR does not signal apoptosis.

The central regulator of the ER, GRP78 [15], is therefore targeted to compensate ER-

stressors of cancerous cells. One simple modification is to shift the dynamic equilibrium

30 of GRP78 expression towards overexpression. Indeed, GRP78 expression is highly

elevated in many cancers, and correlates with malignancy, metastasis, and drug resistance

[83-93]. The elevated GRP78 phenotype is observed in breast cancer [83], hepatocellular carcinomas [94], gastric tumors [95], and oesophageal adenosarcomas [96]. Moreover,

GRP78 is critical to tumor progression as demonstrated by (Lee and her colleagues)

GRP78 inhibition [26]. Taken together, this data begs several questions. Why is GRP78 needed for cancer progression, and possibly metastasis? How does GRP78 overexpression inhibit cell death? Furthermore, what mechanisms upregulate GRP78? It is known that OASIS and XBP-1 upregulate GRP78, could these proteins be responsible?

These questions will be addressed in the following six sections.

6.1. The Role of GRP78 in Cancer Progression and Metastasis

Currently it is not clear how GRP78 contributes to metastasis; however, evidence is emerging that both GRP78 and GRP94 have anti-apoptotic qualities. Indeed, the data point to a protective advantage from both the host defenses and chemotherapeutic drugs

[97-99]. This is consistent with elevated GRP78 cancer cell line-expressers that are resistant to Tumor Necrosis Factor and Killer T Cells. This resistant phenotype is reversed by GRP78 knocked-down with antisense [26]. GRP78 overexpression partly attenuates an UPR [100], suggesting that GRP78 increases the buffering capacity of ER for unfolded protein accumulation. Taken together, these data suggest GRP78 overexpression reduces ER-stress and apoptosis by reducing the effect of toxic agents and the extent of an UPR.

31 6.2. Mechanisms of GRP78 Cell Death Inhibition

In a review authored by Jianze Li and Amy S. Lee in 2006, titled Stress Induction of

GRP78/BiP and Its Role in Cancer this group put forward three possible mechanisms

describing how GRP78 overexpression may inhibit apoptosis and cell death [82]. They

postulated one protective mechanism of GRP78 overexpression may relate to its ability to

buffer calcium in the ER [82]. Indeed, GRP78 itself buffers roughly 25% of ER- sequestered calcium [41]. Their mechanism suggests that calcium reuptake in the ER is coupled to calcium buffering in the mitochondria. Mitochondria generate the majority of

ATP through the Citric Acid Cycle and Electron Transport Chain. Calcium is a cellular signaling molecule that is buffered in both the ER and mitochondria. Calcium buffering within the mitochondria also has a role in apoptotic cell death. Indeed, if mitochondrion experience calcium overload, then they produce oxidants causing oxidative stress possibly triggering apoptosis [101]. Calcium signaling between the mitochondria and the

ER is documented following the generation of nitric oxide (NO). Nitric oxide is a soluble oxidant and signaling molecule which competes with oxygen for cytochrome c binding.

Cytochrome c is a molecule of the Electron Transport Chain. Production of NO inhibits cytochrome c of the Electron Transport Chain evoking mitochondrial calcium fluxes.

These calcium fluxes perturb ER calcium levels and initiate an UPR signaled by both the cleavage of ATF6 and upregulation of GRP78. This study provides the first evidence that NO-dependent disruption of the mitochondria is coupled to ER-stress. It also confirms GRP78 protection against toxic agents, such as excessive NO [102].

32 A second mechanism is that GRP78 may directly inhibit pro-apoptotic signaling proteins of the UPR by directly binding with them [82]. This mechanism draws upon the observation that purified Heat Shock Protein 70 (HSP70 a GRP78 family member) can interact with the cytosolic Apoptotic Protease Activating Factor 1 (apaf1) and procaspase

9 signaling complexes. This interaction inhibits caspase processing. Caspases are a type of protease involved in inflammation and apoptosis. Authors Li and Lee state that

GRP78 inhibition of apoptosis is drawn from analogy of HSP70 inhibiting apoptosis through apaf1/caspase 9 binding. However, this hypothesis is strengthened by the observations that GRP78 coprecipitates with caspase 12 in an in vivo murine model.

Furthermore, recombinant GRP78 inhibits activation of caspase 3 and caspase 12 in cell- free systems [103].

A third mechanism is that elevated GRP78 expression inhibits apoptosis by reducing unfolded and/or misfolded protein accumulation in the ER and by decreasing the intensity

UPR signaling [82]. In other words, GRP78 overexpression may attenuate an UPR in cancerous cells by increasing their ER’s buffering capacity for unfolded proteins and by dampening both the amplitude and duration of the UPR. Therefore, GRP78 overexpression increases the likelihood a cancerous cell would not experience cell death through apoptosis.

6.3. Mechansims of Upregulating GRP78

ATF6, OASIS, and XBP-1 transcriptionally upregulate GRP78 expression [22, 46, 49,

66, 73]. It is striking that all the ER-stress transducers, including OASIS and the XBP-1

33

signaling molecule, are linked with cancer [26, 38, 39, 104, 105]. This suggests several

mechanisms cancer employs to survive ER-stress. First, mutations to these signaling

molecules may inhibit their activation of apoptosis. Second, mutations to these signaling

molecules could increase their induction of ER-chaperone proteins, like GRP78. Third,

perhaps mutations either enhance the expression or half-life of GRP78. Any of these

mechanisms could elevate GRP78 expression and reduce both the intensity and duration

of UPR signaling thus reducing apoptosis.

6.4. The Link between Dedifferentiated Osteoblasts, OASIS, and XBP-1

One mechanism of overexpression could involve osteoblast dedifferentiation. Cell-cycle,

pRB, and RUNX2 signaling are important to osteoblast differentiation [10]. Moreover,

pRB is largely inactivated in osteosaroma [4], and osteosarcoma is considered an

undifferentiated tumor [80, 81]. If the hypotheses stated above is correct, osteoblast

differentiation programming transcriptionally regulates both OASIS and XBP-1

expression, then a less differentiated osteoblast could upregulate OASIS and XBP-1 as part of its early programming. Overexpression of OASIS and XBP-1 may in turn upregulate GRP78 expression. In other words, dedifferentiated osteosarcomas may revert to a prior stage of cellular differentiation that induces high levels of both OASIS and

XBP-1, and these high levels evoke elevated GRP78 expression (see figure 6). This hypothesis is consistent with osteosarcomas producing high amounts of osseous tissue and surviving ER-stress.

34 6.4.1. The Genetic Links between OASIS and GRP78 Overexpression

Underlying chromosomal abnormalities of the OASIS (CREB3L1) gene may facilitate

GRP78 overexpression. A possible catalyst is a genetic translocation such as those

observed in low grade fibromyxoid sarcomas (LGFMS). In a percentage of LGFMS,

chromosomal translocations result in the fusion of the FUS gene with the CREB3L1

(FUS-CREB3L1) gene [37, 106]. OASIS overexpression mediating elevated GRP78

expression is supported with a report that intended to both characterize and distinguish

between mesenchymal cancers. This report demonstrates that osteosarcoma presents

with elevated levels of OASIS [39]. Taken together, perhaps a FUS-CREB3L1 transgene

in osteosarcoma causes OASIS overexpression which may upregulate GRP78 and in turn

reduces UPR-mediated apoptosis.

This hypothesis is supported by a few observations. To begin, chromosomal

abnormalities that cause gains or amplification of the long arm of chromosome 1 are

commonly associated with cancer [107]. Indeed, it known that an amplicon near APOA2

gene in the q21-q23 region of chromosome 1 causes overexpression of ATF6 in human

sarcomas. ATF6 is overexpressed eight-to-nine fold in this cancer. This report advanced

the idea that overexpression of ATF6, because it actively targets UPREs and ERSEs, could elevate GRP78 expression [108]. OASIS also targets UPREs and ERSEs. Other

supportive evidence comes from genetic translocations of FUS with the CHOP gene.

The CHOP gene codes for another protein associated with the UPR and apoptosis. This

particular genetic translocation produces high levels of a FUS-CHOP fusion protein via

the FUS genetic promoter. Overexpression of the FUS-CHOP fusion protein in

35 transgenic mice contributes to their pathogenesis of liposarcomas [109]. Together, this

points to the following hypothesis. A FUS-OASIS transgene may constitutively produce high levels of active OASIS (via the FUS promoter) which may upregulate GRP78 in osteosarcoma.

6.4.2. The Link between XBP-1 and GRP78 Overexpression

The levels of XBP-1 expression in osteosarcoma are currently unknown. However, the links between XBP-1 and cancer are extensive. Indeed, XBP-1 is found to be upregulated and essential to tumor growth under hypoxic conditions which cause ER- stress. In a murine model of cancer growth, XBP-1-null mouse embryonic fibroblasts transplated into other mice, compared to wild-type controls, have severely inhibited tumor growth in response to hypoxic environments [38].

Other studies linking XBP-1 with cancer and tumor progression report similar findings.

For instance, high levels of XBP-1 expression are associated with breast cancer [110], hepatocellular carcinoma [94], and in both colorectal adenomas and adenocarinomas

[111]. However, a contradictory role for XBP-1 is noted. This report used “organ- confined” prostate cancers from patients who underwent total cystoprostatectomy or prostatectomy for bladder and prostate cancers, respectively. These subjects reported

XBP-1 downregulation correlates with an elevated Gleason’s score [112].

A Gleason score is given to a prostate cancer based upon microscopic examination of either biopsy or needle-punch samples and indicates tumor progression. Gleason’s scale

36 is from 1 through 5. Grade 1 resembles a well differentiated and normal prostate tissue

with closely packed and well formed glands. A grade 5 Gleason score resembles poorly

differentiated tissue that does not have recognizable glands and often has sheets of cells

invading surrounding tissue. This report demonstrates XBP-1 in human prostate cancers

is lowest in grade 1, peaks at grade 3, and diminishes in grade 5 [112]. Therefore, this

report is correct in saying that advanced prostate cancers are negatively associated with

XBP-1 expression. However, their title is misleading because XBP-1 expression peaks during tumor progression. More importantly, this report does not address this transient

XBP-1 peak during prostate cancer maturation. Clearly some interesting cancer biology involves elevated XBP-1.

This is consistent with what researcher Albert Koong and associates report; XBP-1 is overexpressed in cancer. In their review, Targeting XBP-1 as a Novel Anti-Cancer

Strategy [113] they described unpublished data using an antibody that targets active or spliced (S) XBP-1. According to them, resected pancreatic adenocarinomas display elevated (S) XBP-1 activity within the tumor. They also pointed out that enhanced XBP-

1 activity is “starkly contrasted” in adjacent “normal” cells of the pancreas; these cells have low XBP-1 activity [113]. This data is consistent with a role for elevated XBP-1 activity promoting cancer progression.

The links between XBP-1 overexpression and cancer, transient or otherwise, are extensive. Therefore, the simplest model of how XBP-1 contributes to cancer is XBP-1 is overexpressed at some developmental cancer stage and its activity assists UPR

37 attenuation in cancer. This attenuation may occur by increasing the volume of the ER

which would reduce the concentration of unfolded proteins within it. Also, enhanced

XBP-1 expression may induce GRP78 expression. Therefore, it is my hypothesis that

osteosarcoma has high levels of XBP-1 at some point during its development.

Little is known regarding XBP-1 genetic or chromosomal abnormalities; however, ATF6

activity induces XBP-1 expression [22, 49]. By analogy, perhaps OASIS overexpression

induces high levels of XBP-1. Another hypothesis, advanced above, perhaps

dedifferentiated osteosarcoma cells revert to an earlier stage of osteoblast signaling which

induces XBP-1 expression. If pRB signaling is inactivated in osteosarcoma and RUNX2-

pRB interactions are required for late stage osteoblast differentiation, then RUNX2

signaling may mediate this early differentiation signaling. If RUNX2 signaling does

induce OASIS and XBP-1 induction early in osteoblast differentiation, then deregulated

RUNX2 signaling in undifferentiated osteoblasts may constitutively overexpress both

XBP-1 and OASIS (see figure 6).

7. Conclusions

In conclusion, these data support a role for OASIS, XBP-1, and UPR in both osteoblast differentiation and osteosarcoma. Four core pieces of evidence point to these roles for

OASIS and XBP-1. First, the primary function of osteoblasts is the formation of bone by

their secretion of extracellular osteoid matrix protein. Second, OASIS and XBP-1 are

concurrently expressed, along with osteopontin, during osteoblast differentiation. Third,

both OASIS and XBP-1 participate in a quality control response to protein folding within

38 the ER called the UPR. Fourth, both OASIS and XBP-1 are linked with cancer, and

OASIS is directly linked to osteosarcoma.

Regarding osteoblast differentiation, the data herein support two hypotheses regarding

OASIS and XBP-1 regulation during osteoblast differentiation. First, both OASIS and

XBP-1 may be upregulated in response to a series of low-level UPRs, insufficient to

initiate apoptosis, as a consequence of progressive osteoid matrix synthesis in maturing

osteoblasts. Second, OASIS and XBP-1 expression may be upregulated through

osteoblast differentiation pathways.

The data reported within this text also support two hypotheses for OASIS and XBP-1

increasing the ER’s protein through-put during osteoblast differentiation. First, XBP-1 activity could elevate the level of protein processing within an osteoblast’s ER by increasing the volume of this compartment and upregulating GRP78. Second, OASIS

within the ER ensures soluble proteins, such as proteoglycans, fold and clear the ER en-

route to the extracellular space.

Concerning osteosarcoma, the role of OASIS and XBP-1 is less clear. Yet it is known

that OASIS is upregulated in osteosarcoma. These data herein support several

hypotheses for the roles of OASIS and XBP-1 in osteosarcoma. With reference to XBP-

1, its levels in osteosarcoma are currently unknown. However, all XBP-1 cancer studies

support high levels of XBP-1 activity at some point during cancer maturation. Perhaps

XBP-1 is highly expressed at a developmental stage of osteosarcoma. If true to

39 osteosarcoma, then perhaps a series of low-level UPRs, initiated by undifferentiated

osteosarcoma-mediated osseous tissue synthesis, insufficient to induce apoptosis, could

upregulate XBP-1 and OASIS. With reference to OASIS, a genetic translocation of the

CREB3L1 gene with the FUS gene, such as those found in LGFMS, may occur in

osteosarcoma causing OASIS overexpression. Lastly, if OASIS and XBP-1 are both transcriptionally regulated by an osteoblast differentiation program, then an

undifferentiated osteosarcoma could evoke their upregulation. Whichever the mechanism

of elevating OASIS and XBP-1 expression, these heightened levels could induce higher

levels of GRP78 and attenuate a previously lethal UPR signaling event.

8. Future Directions

It is evident more research is needed to detail the molecular mechanisms promoting

osteoblast differentiation and osteosarcoma. However, the hypotheses presented here

may provide key mechanistic insight.

An important experiment would be to determine whether the UPR is critical to both

osteoblast differentiation and osteosarcoma by inhibiting GRP78. If GRP78 inhibition in

maturing osteoblasts resulted in extensive cell death, or reduced the level of osteoblast

markers, then this would support the hypothesis of differentiating osteoblasts scaling-up

protein processing through the ER for high-level secretion. If GRP78 inhibition on

osteosarcoma models resulted in either increased cell death or reduced cell growth, then

this would support the hypothesis that osteosarcomas use elevated GRP78 expression to

reduce a once lethal UPR to sublethal levels. If GRP78 inhibition attenuates osteoblast

40 differentiation and osteosarcoma, then mechanistic of promoting GRP78 expression should be tested.

Before exploring mechanistic GRP78 promoting experiments, a simple way of inhibiting

GRP78 will be discussed. Inhibitory techniques include genetic manipulations and pharmacologic inhibition. However, genetic modifications are time consuming. On the other hand, pharmacological experiments have poor specificity, but poor specificity is a trade-off with quick proof-of-principle experimentation. Versipelostatin (VST) is a pharmacological inhibitor of GRP78 expression. VST physically binds with ERSEs within the GRP78 gene to inhibit its expression. This compound, along with two others

(pyrisulfoxin and alternariol), were identified as inhibitors in a screening assay using an

ERSE-luciferase reporting vector. ERSEs are present within the promoter regions of the

GRP78, GRP94, and calreticulin genes. This suggests that VST may inhibit their expression as well. However, VST is currently only known to inhibit GRP78 expression which it does strongly [22, 114-116].

To determine whether osteoblast differentiation signaling induces OASIS and XBP-1 expression, known osteoblast differentiation inducers should be applied to an osteoblast differentiation model (for instance the hFOB 1.19 cell culture model). If OASIS and

XBP-1 expression is increased, then this supports the mechanism of an osteoblast differentiation program inducing OASIS and XBP-1. Further studies should determine whether RUNX2 or Osterix activity cause OASIS and XBP-1 expression. This mechanism could be probed by inhibiting RUNX2 or Osterix activity after osteoblast

41 differentiation is induced while measuring OASIS and XBP-1 expression. Another

experiment to probe this mechanism could use a RUNX2 or Osterix overexpression

system while measuring OASIS and XBP-1 induction. Moreover, identifying a promoter

region within the OASIS and XBP-1 genes targeted by RUNX2 or Osterix would strengthen a mechanistic relationship, especially if RUNX2 or Osterix proteins bound to

OASIS or XBP-1 genetic elements could be isolated.

To test the whether a (or series of) sublethal UPR(s) promote osteogenic commitment could be determined with an osteoblast differentiation model. Using this model, a pulse

(or series of pulses) of ER stressors could be administered. Then, a time course of

OASIS, XBP-1, and GRP78 induction should be measured along with osteoblast markers.

This experiment would help determine whether OASIS, XBP-1, and GRP78 expression are increased in response to a sublethal UPR. Also, this experiment would identify whether a sublethal UPR promotes osteogenic commitment.

To determine whether GRP78 upregulation is induced by OASIS and XBP-1 in maturing osteoblasts, again an osteoblast differentiation model could be employed. First, mutations could be introduced to OASIS and XBP-1 overexpression vectors that prevent their activation (target mutation sites are outlined in sections 3.1.4 and 3.1.5). Then, either vector (or both together) could be applied to differentiating osteoblasts to determine whether OASIS and/or XBP-1 activity reduces GRP78 levels and osteoblast markers by comparison to controls.

42 Other ways to begin probing the hypotheses reported within this text would be to measure

OASIS, XBP-1, and GRP78 expression levels in osteosarocoma cell culture models. A hypothesis above predicts a correlation between high OASIS and XBP-1 expression with

high GRP78 expression levels. If true, then other experiments should determine whether elevated OASIS and XBP-1 levels increase GRP78 expression in these models. This experiment could be done using the previously tested osteosarcoma cells and knocking down their OASIS or XBP-1 expression while measuring GRP78 expression. This experiment could also be done by using overexpression vectors that have mutations which inhibit OASIS and XBP-1 activity.

Other experiments should determine whether an underlying genetic mutation, such as a genetic translocation of the FUS gene with the CREB3L1 gene, occurs in osteosarcoma.

Identifying genetic muations within the CREB3L1 gene could be done by isolating mRNA transcripts from osteosarcoma cell culture models, primary tumors, or paraffin- fixed tumor specimens. After mRNA is isolated and processed it can sequenced or probed with primers targeting the FUS-CREB3L1 genetic translocation [37, 106]. If a mutation is identified within the OASIS gene, then this mutation should be tested to identify whether it overexpresses a functional OASIS protein. Furthermore, if an OASIS transgene or mutation is identified, then it should be determined whether this mutation within induces GRP78 expression and to what extent this mutation contributes to the pathogenesis of osteosarcoma. The first experiment could be done by overexpressing an identified mutation within OASIS using an osteoblast cell culture model while measuring

43 GRP78. The second experiment could be done by introducing an OASIS mutation into a

transgenic mouse and measuring the incidence of osteosarcoma.

Further experiments could use the pharmacological inhibitor of S1P (AEBSF) to identify

to what extent OASIS cleavage fragments (p55 and p50) contribute to osteoblast

differentiation or osteosarcoma. As stated in section 3.1.5 OASIS p55 and p50 induce

GRP78 at different levels. Perhaps the reason these fragments induce GRP78 at different

levels is because they localize to different cellular compartments. Perhaps OASIS p55

has a localization motif specific to the nucleus whereas OASIS p50 has a motif specific

to localization in the cytoplasm. These different motifs could be the result of S1P versus

S2P cleavage of OASIS. Another possibility behind OASIS p55 and p50 differential

GRP78 induction levels is that one of these fragments may interact with the bZIP domain

of XBP-1. The OASIS and XBP-1 bZIP interaction theory was advanced by researchers

Toshio Nikaido and colleagues [34]. Perhaps one of the two OASIS cleavage fragments

binds to XBP-1’s bZIP domain higher than the other. Perhaps this higher binding

elevates the levels at which the cleaved OASIS bZIP motif, together with active XBP-1,

induces GRP78.

Still other experiments that examine the differences between OASIS p55 and p50 should

examine whether either cleavage fragment is a stronger promoter of osteoblast

differentiation. These experiments could be accomplished by using expression systems

that only express either the OASIS p55 or p50. Applying one of these expression

systems to an osteoblast differentiation model while measuring osteoblast markers would

determine the extent each cleavage fragment contributes to the maturation process.

44

Additional, future directions should determine whether OASIS contains a domain that

regulates GRP78 binding to it ER luminal domain, like ATF6. This experiment could be accomplished by examining OASIS and ATF6 similarities within their luminal domains.

Supportive experiments should attempt to isolate the OASIS-GRP78 complex without the use of cross-linking agents. If possible, then isolating this complex would support

OASIS’s active regulation of GRP78 binding. In addition, if my hypothesis is correct

about OASIS promoting secretion of proteoglycans from the ER, then OASIS knock-out

mice should have elevated proteoglycans levels within their ER. Furthermore, if OASIS

does restart the GRP78 ATPase cycle, then mutating this lumenal site should accomplish

two things. Firstly, proteins should accumulate within the ER of differentiating

osteoblasts, similar to the OASIS knock-out mouse. Secondly, less proteoglycans will be

secreted by differentiating osteoblasts because they are not leaving the ER.

45 Cell Cycle P P P P pRB

G2 G1 P P S pRB

P Vitamin D P BMP Ascoribic Acid pRB Notch Signaling RUNX2

RUNX2 Osterix

MSC Osteoprogenitors Committed Osteoblasts Early Osteoblasts Mature Osteoblasts Quiescent Activated/ Proliferating

Self-generating MSC population Figure 1 | Osteoblast differentiation signaling: from mesenchymal stem cells (MSCs) to mature osteoblasts. This figure is adapted from Deshpande 2006. This figure portrays how the cell cycle, Retinoblastoma Protein (pRB), RUNX2, and Osterix signaling may interact to induce osteoblast differentiation. Vitamin D, Bone

Morphogenic Protein (BMP), Ascorbic Acid, and Notch Signaling induce RUNX2 expression in MSCs. RUNX2 expression drives MSCs to become osteoprogenitors. If

Osterix is induced, then there is osteogenic-commitment. The pRB signaling molecule is involved in cell cycle signaling. If pRB becomes hypo-phosphorylated, then it physically interacts with RUNX2, and may signal the final stages of osteoblast differentiation.

46

Degradation Transport Vesicle To the Golgi (Targeted for Secretion) Proteasome

GRP78 Unfolded/ Folded Misfolded Protein Protein Ca2+ Disulfide ER Stress Isomerase Ca2+ Transducers: 2+ OASIS, ATF6, GRP94 ATP Ca IRE1, PERK Highly GRP78 Branched 2+ Glycan Sugars Ca Nascent Ca2+ O2 Protein Calnexin Unfolded Protein ER Lumen Response ER Membrane Cytosol Figure 2 | The pathways within the endoplasmic reticulum (ER) that assist protein quality control. This figure is partially adapted from Ma and Hendershoot, 2004. This figure depicts an influx of proteins entering the ER. These proteins are assisted by various enzymes and cofactors to fold properly. Once the protein reaches a thermodynamically stable conformation it is considered folded. Proteins are either targeted to various cellular compartments, transmembrane locations, or for secretion. If the protein becomes spontaneously denatured (unfolded) or is misfolded, then these proteins are refolded by chaperone proteins (like GRP78) or are cleared from the organelle. If unfolded proteins accumulate, then this stress is perceived by the ER-stress transducers (OASIS, ATF6, IRE1, and PERK) and signaled as an Unfolded Protein

Response (UPR). GRP78 dissociation from these ER-stress transducers is believed to buffer unfolded protein accumulation.

47 ER Stress

PERK IRE1 ATF6 OASIS N Unfolded N Unfolded C C Proteins Proteins Unfolded Proteins ER Lumen P P Cytosol P P RIP: C NNS1P & S2P C AAA

AAA eIF2α eIF2α P pATF6(N) pOASIS(N) p55 and p50

Unspliced Transcriptional XBP-1 Attenuation peptide Spliced XBP-1 peptide Translational Induction of ATF4 Unknown Function ERSE/UPRE ER Chaperones, XBP1, etc.

Figure 3 | Endoplasmic reticulum (ER) stress and Unfolded Protein Response

signaling. This diagram is partially adapted from Kazutoshi Mori, 2003. This model depicts the stress transducers of the ER: PERK, IRE1, ATF6, and OASIS. Also included

in the diagram are eIF2α and XBP-1, signaling molecules of the UPR. When GRP78 is

recruited away from PERK and IRE1 these proteins form homodimers and they become

active giving rise to the above signaling. It is thought that GRP78 interacts with ATF6

differently then it does with PERK and IRE1, perhaps this unique interaction also occurs

between OASIS and GRP78. Regardless, ER stress also induces proteolytic

modifications to OASIS and ATF6 by a Regulated Intramembrane Proteolysis (RIP)

mechanism which cleaves membrane-bound transcriptions factors.

48 49 N IRE1 Unfolded Proteins ER Lumen

Cytosol P P Stem-loop structures of unspliced XBP-1 mRNA are targeted splice sites of activated IRE1 C

Unspliced XBP1 mRNA AAA

AUG 26 nt UAA Spliced

Spliced XBP1 mRNA

AAA

AUG Reading Frame Shift UAA

Figure 4 | Frame switch splicing of XBP-1 by activated IRE1. This diagram is partially adapted from Kazutoshi Mori, 2003. This figure depicts how IRE1 forms a homodimer after unfolded proteins accumulate and mediates frame switch splicing of

XBP-1. Unspliced XBP-1 mRNA codes for a protein with an unknown function when translated. When IRE1 is activated by unfolded proteins, this active homodimer targets the stem loop structure of XBP-1 mRNA and removes 26 nucleotides from this mRNA transcript. The two pieces of mRNA, which flank the 26 nucleotide fragment, are covalently bound together by a tRNA ligase. This splicing produces a reading frame shift. This reading frame shift, or frame switch splicing (as coined by Mori), introduces an entirely new protein after the splice site. In this case, frame switch splicing regulates a potent XBP-1 transcription factor.

50

P Vitamin D P BMP Ascorbic Acid pRB Notch Signaling RUNX2

RUNX2 Osterix

Activated/ Quiescent Proliferating

MSC Osteoprogenitors Committed Osteoblasts Early Osteoblasts Mature Osteoblasts

? Osteocalin Collagen type I IA Alkaline Phosphotase Osteopontin

OASIS XBP-1 Promoter Protein Expression Level Profile Element ? DNA

Figure 5 | A model of osteoblast differentiation signaling inducing OASIS and XBP-

1. This figure depicts how RUNX2 and Osterix signaling pathways are thought to

mediate transcription of osteoid matrix proteins: Collagen type 1 IA, Alkaline

Phosphotase, and Osteopontin. This figure also shows how late stage Retinoblastoma protein (pRB) interactions with RUNX2 are thought to induce Osteocalcin expression during osteoblast differentiation. Further, this figure depicts a hypothetical pathway used

by maturing osteoblasts that induces transcription of OASIS and XBP-1. Hypothetically,

a mechanism such as this could enable OASIS and XBP-1 proteins to prepare the ER for

synthesized proteins destined to become osseous tissue.

51 P Vitamin D P BMP Ascoribic Acid pRB Notch Signaling RUNX2

RUNX2 Osterix Activated/ ? Quiescent Proliferating

MSC Osteoprogenitors Committed Osteoblasts Early Osteoblasts Mature Osteoblasts

? Osteocalin Collagen type I IA

Alkaline Phosphotase

Osteopontin

OASIS

XBP-1 Promoter Protein Expression Level Profile Element ? DNA Figure 6 | A hypothetical mechanism of Retinoblastoma Protein (pRB) inactivation

conferring increases in OASIS and XBP-1 expression. If the hypothesis advanced

here regarding osteoblast differentiation signaling inducing OASIS and XBP-1 transcription are correct, then inactivation of pRB signaling could cause mature osteoblasts to revert to an earlier stage of differentiation. This type of signaling would be beneficial to a cancerous cell because it would protect against cell death by increasing the cells resistance to ER stressors (see test for details). This model would also be consistent with the data suggesting that osteosarcomas are dedifferentiated osteoblasts secreting osseous tissue.

52 References

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