Transcriptome analysis of polyploid giant cancer cells and their progeny reveals a functional role for p21 in polyploidization and depolyploidization.

Polyploid giant cancer cells (PGCC) are frequently detected in tumors and are increasingly recognized for their roles in chromosomal instability and associated genome evolution that leads to cancer recurrence. We previously reported that therapy stress promotes polyploidy, and that acid ceramidase plays a role in depolyploidization. In this study, we used an RNA-seq approach to gain a better understanding of the underlying transcriptomic changes that occur as cancer cells progress through polyploidization and depolyploidization. Our results revealed gene signatures that are associated with disease-free and/or overall survival in several cancers and identified the cell cycle inhibitor CDKN1A/p21 as the major hub in PGCC and early progeny. Increased expression of p21 in PGCC was limited to the cytoplasm. We previously demonstrated that the sphingolipid enzyme acid ceramidase is dispensable for polyploidization upon therapy stress but plays a crucial role in depolyploidization. The current study demonstrates that treatment of cells with ceramide is not sufficient for p53-independent induction of p21 and that knockdown of acid ceramidase, which hydrolyzes ceramide, does not interfere with upregulation of p21. In contrast, blocking the expression of p21 with UC2288 prevented the induction of acid ceramidase and inhibited both the formation of PGCC from parental cells as well as the generation of progeny from PGCC. Taken together, our data suggest that p21 functions upstream of acid ceramidase and plays an important role in polyploidization and depolyploidization.

Polyploid giant cancer cells are dependent on cholesterol for progeny formation through amitotic division.

Polyploid Giant Cancer Cells (PGCC) are increasingly being recognized as drivers of cancer recurrence. Therapy stress promotes the formation of these cells, which upon stress cessation often successfully generate more aggressive progeny that repopulate the tumor. Therefore, identification of potential PGCC vulnerabilities is key to preventing therapy failure. We have previously demonstrated that PGCC progeny formation depends on the lysosomal enzyme acid ceramidase (ASAH1). In this study, we compared transcriptomes of parental cancer cells and PGCC in the absence or presence of the ASAH1 inhibitor LCL521. Results show that PGCC express less INSIG1, which downregulates cholesterol metabolism and that inhibition of ASAH1 increased HMGCR which is the rate limiting enzyme in cholesterol synthesis. Confocal microscopy revealed that ceramide and cholesterol do not colocalize. Treatment with LCL521 or simvastatin to inhibit ASAH1 or HMGCR, respectively, resulted in accumulation of ceramide at the cell surface of PGCC and prevented PGCC progeny formation. Our results suggest that similarly to inhibition of ASAH1, disruption of cholesterol signaling is a potential strategy to interfere with PGCC progeny formation.

Autophagy modulating therapeutics inhibit ovarian cancer colony generation by polyploid giant cancer cells (PGCCs).

BACKGROUND: Genomic instability and chemoresistance can arise in cancer due to a unique form of plasticity: that of polyploid giant cancer cells (PGCCs). These cells form under the stress of chemotherapy and have higher than diploid chromosome content. PGCCs are able to then repopulate tumors through an asymmetric daughter cell budding process. PGCCs have been observed in ovarian cancer histology, including the deadly and common form high-grade serous ovarian carcinoma (HGSC). We previously discovered that drugs which disrupt the cellular recycling process of autophagy are uniquely efficacious in pre-clinical HGSC models. While autophagy induction has been associated with PGCCs, it has never been previously investigated if autophagy modulation interacts with the PGCC life cycle and this form of tumor cell plasticity. METHODS: CAOV3 and OVCAR3 ovarian cancer cell lines were treated with carboplatin or docetaxel to induce PGCC formation. Microscopy was used to characterize and quantify PGCCs formed by chemotherapy. Two clinically available drugs that inhibit autophagy, hydroxychloroquine and nelfinavir, and a clinically available activator of autophagy, rapamycin, were employed to test the effect of these autophagy modulators on PGCC induction and subsequent colony formation from PGCCs. Crystal violet-stained colony formation assays were used to quantify the tumor-repopulating stage of the PGCC life cycle. RESULTS: Autophagy inhibitors did not prevent PGCC formation in OVCAR3 or CAOV3 cells. Rapamycin did not induce PGCC formation on its own nor did it exacerbate PGCC formation by chemotherapy. However, hydroxychloroquine prevented efficient colony formation in CAOV3 PGCCs induced by carboplatin (27% inhibition) or docetaxel (41% inhibition), as well as in OVCAR3 cells (95% and 77%, respectively). Nelfinavir similarly prevented colony formation in CAOV3 PGCCs induced by carboplatin (64% inhibition) or docetaxel (94% inhibition) as well as in OVCAR3 cells (89% and 80%, respectively). Rapamycin surprisingly also prevented PGCC colony outgrowth (52-84% inhibition). CONCLUSIONS: While the autophagy previously observed to correlate with PGCC formation is unlikely necessary for PGCCs to form, autophagy modulating drugs severely impair the ability of HGSC PGCCs to form colonies. Clinical trials which utilize hydroxychloroquine, nelfinavir, and/or rapamycin after chemotherapy may be of future interest.

Ceramide Synthase 6 Maximizes p53 Function to Prevent Progeny Formation from Polyploid Giant Cancer Cells.

Polyploid giant cancer cells (PGCC) constitute a transiently senescent subpopulation of cancer cells that arises in response to stress. PGCC are capable of generating progeny via a primitive, cleavage-like cell division that is dependent on the sphingolipid enzyme acid ceramidase (ASAH1). The goal of this study was to understand differences in sphingolipid metabolism between non-polyploid and polyploid cancer cells to gain an understanding of the ASAH1-dependence in the PGCC population. Steady-state and flux analysis of sphingolipids did not support our initial hypothesis that the ASAH1 product sphingosine is rapidly converted into the pro-survival lipid sphingosine-1-phosphate. Instead, our results suggest that ASAH1 activity is important for preventing the accumulation of long chain ceramides such as C16-ceramide. We therefore determined how modulation of C16-ceramide, either through CerS6 or p53, a known PGCC suppressor and enhancer of CerS6-derived C16-ceramide, affected PGCC progeny formation. Co-expression of the CerS6 and p53 abrogated the ability of PGCC to form offspring, suggesting that the two genes form a positive feedback loop. CerS6 enhanced the effect of p53 by significantly increasing protein half-life. Our results support the idea that sphingolipid metabolism is of functional importance in PGCC and that targeting this signaling pathway has potential for clinical intervention.

The Uninsured are a Distinct Population in a Breast Cancer Cohort, with Unique Screening, Staging, and Outcomes.

Insurance status is a known determinant of cancer stage at diagnosis and outcome. However, insurance status can change over the course of the disease and its treatment, complicating causal analysis. Cancer registries strive to capture the insurance status of patients at diagnosis, but this is not always possible. Breast cancer poses a particular challenge for this effort, as uninsured patients become eligible for Medicaid upon the diagnosis. Thus, their insurance status may have changed from uninsured to Medicaid by the time registrars interact with treatment records. We addressed this potential blurring between categories by working with a sample of patients identified through the cancer registry of the Medical University of South Carolina to focus on determining insurance status at diagnosis whenever possible. We found that the uninsured population (32 women) was larger than the Medicaid-covered population (22 women) in a sample of patients in South Carolina, a state that did not accept the Medicaid expansion. Compared with women who carried any type of insurance, uninsured women were much more likely to find their own breast mass through palpation rather than through screening, they were diagnosed with a later stage of breast cancer at diagnosis, and their outcomes were worse. Insured women experienced significantly increased survival odds (odds ratio, 3.28) and multiple regression analysis demonstrated that the higher stages seen in uninsured women largely accounted for the poorer outcomes. These findings suggest that more research is needed to define the characteristics and disease courses unique to the breast cancer population lacking insurance prior to diagnosis.

Giants and monsters: Unexpected characters in the story of cancer recurrence.

Polyploid giant cancer cells (PGCC) constitute a dangerous subpopulation of cancer cells and are a driving force in cancer recurrence. These unique cells arise from diploid tumor cells in response to stress encountered in the tumor microenvironment or during cancer therapy. PGCC are greatly dedifferentiated, acquire pluripotency, and are able to replicate through a form of asymmetric division called neosis, which results in new populations that are themselves able to differentiate into new cell types or to re-establish tumors. Progeny tend to be more genetically unstable than the founding population due to the dysregulation required to transition through a PGCC state. Therefore, cancers that escape stressors through this mechanism tend to re-emerge with a more aggressive phenotype that is therapy resistant. This review focuses on the clinical significance of PGCC, the need for standardized nomenclature and molecular markers, as well as possible avenues to develop therapies aimed at PGCC and the process of neosis. The biology underlying the development of PGCC including cell cycle checkpoint dysregulation, stress responses, dedifferentiation, stemness and epithelial-mesenchymal transition is discussed.

An assessment of racial differences in epidemiological, clinical and psychosocial factors among head and neck cancer patients at the time of surgery.

OBJECTIVE: Racial disparities have been well characterized and African American (AA) patients have 30% lower 5-year survival rates than European Americans (EAs) for head and neck squamous carcinoma (HNSCC). This poorer survival can be attributed to a myriad of different factors. The purpose of this study was to characterize AA-EA similarities and differences in sociodemographic, lifestyle, clinical, and psychosocial characteristics in HNSCC patients near the time of surgery. METHODS: Setting: Single tertiary care center. Participants: Thirty-nine newly diagnosed, untreated HNSCC patients (n = 24 EAs,n = 15 AAs) who were to undergo surgery were recruited. Study Design: Cross-sectional study Sociodemographic, lifestyle factors, and disease factors (cancer site, AJCC clinical and pathologic stage, and HPV status)were assessed. Risk factors, leisure time, quality of life and social support were also assessed using validated questionnaires. Exposures: EA and AA patients were similar in the majority of sociodemographic factors assessed. AAs had a higher trend toward pathologically later stage disease compared to EAs and significantly increased time to treatment. RESULTS: EA and AA patients were similar in the majority of sociodemographic factors assessed. AAs had a higher trend toward pathologically later stage disease compared to EAs. AAs also had significantly increased time to treatment (P = 0.05). The majority of AA patients (62%) had later stage pathologic disease. AA were less likely to complete high school or college (P = 0.01) than their EA counterparts. Additionally, AAs were more likely to report having a gap in health insurance during the past decade (37% vs. 15%). CONCLUSIONS: This preliminary study demonstrates a similar profile of demographics, clinical and psychosocial characteristics preoperatively for AAs and EAs. Key differences were AAs tending to have later pathologic stage disease, educational status, delays in treatment initiation, and gaps in health insurance.

Tamoxifen is a candidate first-in-class inhibitor of acid ceramidase that reduces amitotic division in polyploid giant cancer cells-Unrecognized players in tumorigenesis.

Polyploid giant cancer cells (PGCC) represent a poorly understood, small subpopulation of tumor cells that are increasingly being recognized for their critical role in therapy resistance, metastasis, and cancer recurrence. PGCC have the potential to generate progeny through primitive or cleavage-like division, which allows them to evade antimitotic insults. We recently demonstrated that the sphingolipid enzyme acid ceramidase (ASAH1) is required for this process. Since specific ASAH1 inhibitors are not clinically available, we investigated whether tamoxifen, which interferes with ASAH1 function via off-target effects, has a potential clinical benefit independent of estrogen signaling. Our results show that tamoxifen inhibits generation of PGCC offspring in prostate cancer, glioblastoma, and melanoma cells. Analysis of two state-level cancer registries revealed that tamoxifen improves survival outcomes for second, nonbreast cancers that develop in women with early stage breast cancer. Our results suggest that tamoxifen may have a clinical benefit in a variety of cancers that is independent of estrogen signaling and could be due to its inhibition of acid ceramidase. Thus the distinct application of tamoxifen as potentially a first-in-class therapeutic that inhibits the generation of PGCC offspring should be considered in future clinical trials.

A Rapid Array-Based Approach to N-Glycan Profiling of Cultured Cells.

Typically, N-glycosylation studies done on cultured cells require up to millions of cells followed by lengthy preparation to release, isolate, and profile N-glycans. To overcome these limitations, we report a rapid array-based workflow for profiling N-glycan signatures from cells, adapted from imaging mass spectrometry used for on-tissue N-glycan profiling. Using this approach, N-glycan profiles from a low-density array of eight cell chambers could be reported within 4 h of completing cell culture. Approaches are demonstrated that account for background N-glycans due to serum media. Normalization procedures are shown. The method is robust and reproducible, requiring as few as 3000 cells per replicate with a 3-20% coefficient of variation to capture label-free profiles of N-glycans. Quantification by stable isotopic labeling of N-glycans in cell culture is demonstrated and adds no additional time to preparation. Utility of the method is demonstrated by measurement of N-glycan turnover rates due to induction of oxidative stress in human primary aortic endothelial cells. The developed method and ancillary tools serve as a foundational launching point for rapid profiling of N-glycans ranging from high-density arrays down to single cells in culture.

Genetic and pharmacological inhibition of acid ceramidase prevents asymmetric cell division by neosis.

Radiation treatment failure or relapse after initial response to chemotherapy presents significant clinical challenges in cancer patients. Escape from initial courses of treatment can involve reactivation of embryonic developmental stages, with the formation of polynuclear giant cancer cells (PGCCs). This strategy of dedifferentiation can insulate cancer cells from a variety of treatments and allows a residual subpopulation to reestablish tumors after treatment. Using radiation or docetaxel chemotherapy, we generated PGCCs from prostate cancer cells. Here, we show that expression of acid ceramidase (ASAH1), an enzyme in the sphingolipid pathway linked to therapy resistance and poor outcomes, is elevated in PGCCs. Targeting ASAH1 with shRNA or treatment with the ASAH1 inhibitor, LCL-521, did not impair the formation of PGCCs, but prevented the formation of PGCC progeny that arise through an asymmetric cell division called neosis. Similar results were obtained in lung cancer cells that had been exposed to radiation or cisplatin chemotherapy as stressors. In summary, our data suggest that endoreplication occurs independent of ASAH1 while neosis is ASAH1-dependent in both prostate and lung cancer cells. Because ASAH1 knockout is embryonic lethal but not deleterious to adult animals, targeting this enzyme has the potential to be highly specific to cells undergoing the dedifferentiation process to escape cancer treatments. Pharmacological inhibition of ASAH1 is a potentially powerful strategy to eliminate cells that could otherwise serve as seed populations for recurrence.

Expression of the SNAI2 transcriptional repressor is regulated by C16-ceramide.

Ceramide synthase 6 (CerS6) is an enzyme that preferentially generates pro-apoptotic C16-ceramide in the sphingolipid metabolic pathway. Reduced expression of CerS6 has been associated with apoptosis resistance and recent studies point to a role for CerS6 in epithelial mesenchymal transition (EMT). Because cells that undergo EMT are also more resistant to apoptosis, we hypothesized that reduced expression of CerS6 could induce changes that are associated with EMT. We found that shRNA-mediated knockdown of CerS6 increases expression of the EMT transcription factor SNAI2 but not SNAI1 or TWIST. Treatment with C6-ceramide nanoliposomes (CNL) resulted in a preferential increase in C16-ceramide and suppressed SNAI2 transcriptional activation and protein expression. The increase in C16-ceramide following CNL treatment was dependent on CerS activity and occurred even when CerS6 shRNA was expressed. shRNA against CerS5, which like CerS6 preferentially generates C16-ceramide, also decreased transcriptional activation of SNAI2, suggesting a role for C16-ceramide rather than a specific enzyme in the regulation of this transcription factor. While loss of CerS6 has been associated with apoptosis resistance, we found that cells lacking this protein are more susceptible to the effects CNL. In summary, our study identifies SNAI2 as a novel target whose expression can be influenced by C16-ceramide levels. The potential of CNL to suppress SNAI2 expression has important clinical implications, since elevated expression of this transcription factor has been associated with an aggressive phenotype or poor outcomes in several types of solid tumors.

Interdiction of Sphingolipid Metabolism Revisited: Focus on Prostate Cancer.

Sphingolipid metabolism is known to play a role in cell death, survival, and therapy resistance in cancer. Sphingolipids, particularly dihydroceramide and ceramide, are associated with antiproliferative or cell death responses, respectively, and are central to effective cancer therapy. Within the last decade, strides have been made in elucidating many intricacies of sphingolipid metabolism. New information has emerged on the mechanisms by which sphingolipid metabolism is dysregulated during malignancy and how cancer cells survive and/or escape therapeutic interventions. This chapter focuses on three main themes: (1) sphingolipid enzymes that are dysregulated in cancer, particularly in prostate cancer; (2) inhibitors of sphingolipid metabolism that antagonize prosurvival responses; and (3) sphingolipid-driven escape mechanisms that allow cancer cells to evade therapies. We explore clinical and preclinical approaches to interdict sphingolipid metabolism and provide a rationale for combining strategies to drive the generation of antiproliferative ceramides with prevention of ceramide clearance.

Prognostic Factors in Myoepithelial Carcinoma of the Major Salivary Glands.

OBJECTIVES: (1) Identify all cases of myoepithelial carcinoma of the major salivary glands from the National Cancer Data Base (NCDB). (2) Analyze the effect of grade, stage, and regional nodal metastasis on survival in myoepithelial carcinoma of the major salivary glands. STUDY DESIGN: Retrospective review of NCDB. SETTING: Multicenter data pooled from 1998 to 2012 in the NCDB. METHODS: We identified all reported cases of myoepithelial carcinomas of the major salivary glands from the United States from 1998 to 2012 in the NCDB. Clinical parameters were then examined and analyzed for predictors of survival. RESULTS: A total of 473 cases of myoepithelial carcinoma were identified. Of the reported cases, 38.1% were low grade; 26.7%, intermediate grade; and 35.2%, high grade. When presenting stage was examined, 24.4% were stage I; 30.6%, stage II; 22.5%, stage III; 12.2%, stage IVa; 3.0%, stage IVb; and 4.1%, stage IVc. At presentation, 18.7% of patients had regional nodal disease, and 4.5% had distant metastases. The 3- and 5-year survival rates were 73% and 64%, respectively. The presence of nodal disease significantly reduced mean survival time versus those without (64 vs 108 months, P < .001), as did high-grade disease compared with low grade (67 vs 114 months, P < .001) and stage III/IV compared with stage I/II disease (61 vs 118 months, P < .001). CONCLUSIONS: The presence of regional nodal disease, high-grade disease, and advanced stage are predictors of lower survival in myoepithelial carcinoma. Further studies based on types of treatment are warranted.

Sex differences in the MB49 syngeneic, murine model of bladder cancer.

OBJECTIVE: The MB49 syngeneic, murine model of bladder cancer has been widely used for more than 35 years. In humans, bladder cancer is one third as prevalent in women as in men, with a trend toward lower prevalence in parous compared to nulliparous women. Our objective was to determine if the MB49 bladder cancer model reproduces the sex differences observed in humans, and to determine its sensitivity to testosterone and the pregnancy hormone, human chorionic gonadotropin (hCG). METHODS: Male and female C57BL/6 mice were implanted with MB49 murine bladder cancer cells, and observed for tumor growth. MB49 dose responses to hCG and dihydrotestosterone were determined in vitro. RESULTS: MB49 tumor growth was significantly greater in male mice than female mice. Pregnancy did not affect MB49 tumor growth in female mice. MB49 cells did not proliferate in response to hCG in vitro and the functional receptor for gonadotropins was absent. Dihydrotestosterone strongly stimulated growth of MB49 cells in vitro. CONCLUSIONS: The MB49 murine model of bladder cancer reproduced some aspects of the sex differences observed in humans. Our results suggest that testosterone may stimulate MB49 cell proliferation, which may explain the more rapid MB49 tumor growth observed in male mice.

Predictors of Nodal Metastasis in Parotid Malignancies: A National Cancer Data Base Study of 22,653 Patients.

OBJECTIVE: (1) To identify predictors of nodal disease in parotid malignancies using various clinical and pathologic variables. (2) To examine the effect of nodal disease on overall survival (OS) in parotid cancers STUDY DESIGN: Retrospective database review. SETTING: National Cancer Data Base (1998-2012). SUBJECTS AND METHODS: We identified all cases of primary parotid malignancies in the United States between 1998 and 2012 in the National Cancer Data Base. Eight histopathologies, constituting >80% of all cases, were examined for nodal metastasis and survival. RESULTS: We identified 22,653 cases of primary parotid cancer. Eight major histologies were studied, with mucoepidermoid carcinoma (31%), acinic cell carcinoma (18%), adenocarcinoma (14%), and adenoid cystic carcinoma (9%) being most common. Regional nodal disease incidence was 24.4% overall and varied by histopathology. Salivary ductal carcinoma had the highest incidence of both nodal metastasis and occult lymph node metastasis. Overall, N0 patients lived significantly longer than N+ (5-year OS, 79% vs 40%; P < .001). Low-grade disease had significantly better survival than high-grade (5-year OS, 88% vs 69%; P < .001). Occult nodal disease was found in 10.2% and varied by histopathology. CONCLUSION: Regional lymph node metastasis significantly decreases survival in many parotid malignancies. High-grade cancers had higher incidences of regional disease than did low grade. Adenocarcinoma had the highest mortality when regional disease was present. Incidence of occult disease varied by histology, but incidence was <10% for all low-grade disease. High T stage and grade are significant independent predictors of nodal disease for most histopathologies.

Regulated lysosomal exocytosis mediates cancer progression.

Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf (-/-) mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance.

Analysis of the National Cancer Data Base to Describe Treatment Trends in Stage IV Oral Cavity and Pharyngeal Cancers in the United States, 1998-2012.

Treatment recommendations for head and neck cancers have evolved over the last several decades, with a particularly clear shift in 2004 toward use of chemotherapy in late-stage patients. This study examines the national trends in treatment combinations for patients with stage IV oral cavity and pharyngeal cancer between 1998 and 2012 using the National Cancer Data Base (NCDB). Our analysis demonstrates that chemotherapy was widely integrated into the treatment plans for this population following 2004, confirming that recommendations were successfully translated into practice. Stage IV patients treated after this shift in treatment experienced higher 5-year survival rates compared to patients treated prior to the adoption of increased chemotherapy usage. We also examined the patient population for other changes over time and found that smaller primary tumors became more common and that 2 primary sites (base of tongue and tonsil) came to represent a larger percentage of the patient population; these changes may also contribute to a rising survival rate. Patients receiving the recommended trimodal therapy of surgery, radiation, and chemotherapy were found to be more geographically widespread over time, suggesting a penetrance of the recommendations into the medical system across the country.

A primer on molecular biology for certified tumor registrars.

Certified tumor registrars (CTRs) are expected to have expertise in cancer staging, treatment, and patient followup and an overall knowledge of the cancer disease process. As medicine becomes more personalized, the prognosis for individual cancer patients is beginning to include more molecular markers, and CTRs are being asked to record these results along with traditional anatomic information about the disease. Molecular markers, also called biomarkers, are measured using a variety of techniques, including fluorescent in situ hybridization (FISH), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and reverse transcription polymerase chain reaction (RT-PCR). This primer will provide an overview of these techniques so that CTRs can more efficiently search medical records for information and more accurately record these data items into abstracting templates.

Molecular chaperone gp96 is a novel therapeutic target of multiple myeloma.

PURPOSE: gp96 (grp94) is a key downstream chaperone in the endoplasmic reticulum (ER) to mediate unfolded protein response (UPR) and the pathogenesis of multiple myeloma is closely linked to dysregulated UPR. In this study, we aimed to determine the roles of gp96 in the initiation and progression of multiple myeloma in vivo and in vitro. EXPERIMENTAL DESIGN: We generated a mouse model with overexpression of XBP1s and conditional deletion of gp96 in B-cell compartment simultaneously to identify the roles of gp96 in the development of multiple myeloma in vivo. Using a short hairpin RNA (shRNA) system, we silenced gp96 in multiple human multiple myeloma cells and examined the effect of gp96 knockdown on multiple myeloma cells by cell proliferation, cell-cycle analysis, apoptosis assay, immunohistochemistry, and human myeloma xenograft model. The anticancer activity of gp96 selective inhibitor, WS13, was evaluated by apoptosis assay and MTT assay. RESULTS: Genetic deletion of gp96 in XBP1s-Tg mice attenuates multiple myeloma. Silencing of gp96 causes severe compromise in human multiple myeloma cell growth through inhibiting Wnt-LRP-survivin pathway. We also confirmed that knockdown of gp96 decreased human multiple myeloma growth in a murine xenograft model. The targeted gp96 inhibitor induced apoptosis and blocked multiple myeloma cell growth, but did not induce apoptosis in pre-B leukemic cells. We have demonstrated that myeloma growth is dependent on gp96 both genetically and pharmacologically. CONCLUSIONS: gp96 is essential for multiple myeloma cell proliferation and survival, suggesting that gp96 is a novel therapeutic target for multiple myeloma. Clin Cancer Res; 19(22); 6242-51. ©2013 AACR.

The role of endoplasmic reticulum stress in maintaining and targeting multiple myeloma: a double-edged sword of adaptation and apoptosis.

Increased cellular protein production places stress on the endoplasmic reticulum (ER), because many of the nascent proteins pass through the ER for folding and trafficking. Accumulation of misfolded proteins in the ER triggers the activation of three well-known pathways including IRE1 (inositol requiring kinase 1), ATF6 (activating transcription factor 6), and PERK (double stranded RNA-activated protein kinase-like ER kinase). The activity of each sensor modulates the overall ER strategy for managing protein quality control as cellular needs change due to growth, differentiation, infection, transformation, and host of other possible physiological states. Here we review the role of ER stress in multiple myeloma (MM), an incurable plasma cell neoplasm. MM is closely linked to dysregulated unfolded protein response in the ER due to the heightened production of immunoglobulin and the metabolic demands of malignant uncontrolled proliferation. Together, these forces may mean that myeloma cells have an "Achilles heel" which can be exploited as a treatment target: their ER stress response must be constitutively active at a remarkably high level to survive their unique metabolic needs. Therefore, inhibition of the ER stress response is likely to injure the cells, as is any further demand on an already over-worked system. Evidence for this vulnerability is summarized here, along with an overview of how each of the three ER stress sensors has been implicated in myeloma pathogenesis and treatment.