Cancer SLC43A2 alters T cell methionine metabolism and histone methylation.

Abnormal epigenetic patterns correlate with effector T cell malfunction in tumours1-4, but the cause of this link is unknown. Here we show that tumour cells disrupt methionine metabolism in CD8+ T cells, thereby lowering intracellular levels of methionine and the methyl donor S-adenosylmethionine (SAM) and resulting in loss of dimethylation at lysine 79 of histone H3 (H3K79me2). Loss of H3K79me2 led to low expression of STAT5 and impaired T cell immunity. Mechanistically, tumour cells avidly consumed methionine and outcompeted T cells for methionine by expressing high levels of the methionine transporter SLC43A2. Genetic and biochemical inhibition of tumour SLC43A2 restored H3K79me2 in T cells, thereby boosting spontaneous and checkpoint-induced tumour immunity. Moreover, methionine supplementation improved the expression of H3K79me2 and STAT5 in T cells, and this was accompanied by increased T cell immunity in tumour-bearing mice and patients with colon cancer. Clinically, tumour SLC43A2 correlated negatively with T cell histone methylation and functional gene signatures. Our results identify a mechanistic connection between methionine metabolism, histone patterns, and T cell immunity in the tumour microenvironment. Thus, cancer methionine consumption is an immune evasion mechanism, and targeting cancer methionine signalling may provide an immunotherapeutic approach.

Autophagic adaptation to oxidative stress alters peritoneal residential macrophage survival and ovarian cancer metastasis.

Tumor-associated macrophages (TAMs) affect cancer progression and therapy. Ovarian carcinoma often metastasizes to the peritoneal cavity. Here, we found 2 peritoneal macrophage subsets in mice bearing ID8 ovarian cancer based on T cell immunoglobulin and mucin domain containing 4 (Tim-4) expression. Tim-4+ TAMs were embryonically originated and locally sustained while Tim-4- TAMs were replenished from circulating monocytes. Tim-4+ TAMs, but not Tim-4- TAMs, promoted tumor growth in vivo. Relative to Tim-4- TAMs, Tim-4+ TAMs manifested high oxidative phosphorylation and adapted mitophagy to alleviate oxidative stress. High levels of arginase-1 in Tim-4+ TAMs contributed to potent mitophagy activities via weakened mTORC1 activation due to low arginine resultant from arginase-1-mediated metabolism. Furthermore, genetic deficiency of autophagy element FAK family-interacting protein of 200 kDa resulted in Tim-4+ TAM loss via ROS-mediated apoptosis and elevated T cell immunity and ID8 tumor inhibition in vivo. Moreover, human ovarian cancer-associated macrophages positive for complement receptor of the immunoglobulin superfamily (CRIg) were transcriptionally, metabolically, and functionally similar to murine Tim-4+ TAMs. Thus, targeting CRIg+ (Tim-4+) TAMs may potentially treat patients with ovarian cancer with peritoneal metastasis.

Phase II clinical trial of metformin as a cancer stem cell-targeting agent in ovarian cancer.

BACKGROUNDEpidemiologic studies suggest that metformin has antitumor effects. Laboratory studies indicate metformin impacts cancer stem-like cells (CSCs). As part of a phase II trial, we evaluated the impact of metformin on CSC number and on carcinoma-associated mesenchymal stem cells (CA-MSCs) and clinical outcomes in nondiabetic patients with advanced-stage epithelial ovarian cancer (EOC).METHODSThirty-eight patients with stage IIC (n = 1)/III (n = 25)/IV (n = 12) EOC were treated with either (a) neoadjuvant metformin, debulking surgery, and adjuvant chemotherapy plus metformin or (b) neoadjuvant chemotherapy and metformin, interval debulking surgery, and adjuvant chemotherapy plus metformin. Metformin-treated tumors, compared with historical controls, were evaluated for CSC number and chemotherapy response. Primary endpoints were (a) a 2-fold or greater reduction in aldehyde dehydrogenase-positive (ALDH+) CD133+ CSCs and (b) a relapse-free survival at 18 months of more than 50%.RESULTSMetformin was well tolerated. Median progression-free survival was 18.0 months (95% CI 14.0-21.6) with relapse-free survival at 18 months of 59.3% (95% CI 38.6-70.5). Median overall survival was 57.9 months (95% CI 28.0-not estimable). Tumors treated with metformin had a 2.4-fold decrease in ALDH+CD133+ CSCs and increased sensitivity to cisplatin ex vivo. Furthermore, metformin altered the methylation signature in CA-MSCs, which prevented CA-MSC-driven chemoresistance in vitro.CONCLUSIONTranslational studies confirm an impact of metformin on EOC CSCs and suggest epigenetic change in the tumor stroma may drive the platinum sensitivity ex vivo. Consistent with this, metformin therapy was associated with better-than-expected overall survival, supporting the use of metformin in phase III studies.TRIAL REGISTRATIONClinicalTrials.gov NCT01579812.

The double edge sword of John Henryism: Impact on patients' health in the People's Republic of China.

The People's Republic of China has experienced extraordinary economic growth, which is associated with increases in chronic health stressors. We examined the impact of John Henryism-a coping mechanism-on various health indicators in a sample of patients (n = 642) in China. John Henryism significantly related to increased medical adherence (B=.03,p<.001) and health-promotional behaviors (B=.02,p<.001). John Henryism predicted several indicators of psychological health through social support. John Henryism was also related to increased alcoholism (B=.04,p<.05). The findings highlight the complexity and paradoxical implications of John Henryism on health. Implications are discussed in relation to China's epidemiological and age demographic shifts.

Oxidative stress controls regulatory T cell apoptosis and suppressor activity and PD-L1-blockade resistance in tumor.

Live regulatory T cells (Treg cells) suppress antitumor immunity, but how Treg cells behave in the metabolically abnormal tumor microenvironment remains unknown. Here we show that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abolish spontaneous and PD-L1-blockade-mediated antitumor T cell immunity. Biochemical and functional analyses show that adenosine, but not typical suppressive factors such as PD-L1, CTLA-4, TGF-ß, IL-35, and IL-10, contributes to apoptotic Treg-cell-mediated immunosuppression. Mechanistically, apoptotic Treg cells release and convert a large amount of ATP to adenosine via CD39 and CD73, and mediate immunosuppression via the adenosine and A2A pathways. Apoptosis in Treg cells is attributed to their weak NRF2-associated antioxidant system and high vulnerability to free oxygen species in the tumor microenvironment. Thus, the data support a model wherein tumor Treg cells sustain and amplify their suppressor capacity through inadvertent death via oxidative stress. This work highlights the oxidative pathway as a metabolic checkpoint that controls Treg cell behavior and affects the efficacy of therapeutics targeting cancer checkpoints.

Gynecologic melanomas: A clinicopathologic and molecular analysis.

OBJECTIVE: Melanoma originating from gynecologic sites (MOGS), including the vulva, vagina, and cervix, is a rare and aggressive form of melanoma with poor long-term clinical outcome. The clinicopathologic features of vulvar and non-vulvar tumors remain relatively understudied, and in contrast to cutaneous melanomas at non-sun-exposed sites, MOGS typically do not harbor BRAF mutations. Thus, we sought to analyze the clinicopathologic and molecular features of MOGS. METHODS: A large retrospective cohort of patients with MOGS (n=59) at a single large academic institution over a 28-year period was identified. Associations among clinicopathologic characteristics were assessed via standard statistical approaches, and clinical outcome was examined using Cox regression analysis. Sanger sequencing was utilized to identify mutations in hotspot regions of BRAF, KIT, NRAS, and CTNNB1. RESULTS: Tumors involving the vagina and/or cervix (non-vulvar) are significantly associated with high-risk clinicopathologic features, including increased tumor thickness, ulceration, positive resection margins, lymph node metastasis, and poor long-term clinical outcome (with increased risk of death due to disease). The aggressive clinical behavior of non-vulvar tumors is independent of advanced clinical stage and lymph node metastasis in multivariate analysis. Targeted molecular analysis confirms an overall low rate of oncogenic mutations in our MOGS cohort, although KIT mutations (particularly in exon 11) are relatively enriched. CONCLUSIONS: Overall, our results show that non-vulvar MOGS are aggressive tumors with poor long-term clinical outcome and indicate that few targeted therapeutic options are currently available to patients with MOGS.

Incidence and Timing of Thromboembolic Events in Patients With Ovarian Cancer Undergoing Neoadjuvant Chemotherapy.

OBJECTIVE: To identify the incidence and timing of venous thromboembolism as well as any associated risk factors in patients with ovarian, fallopian tube, or primary peritoneal cancer undergoing neoadjuvant chemotherapy. METHODS: We conducted a retrospective cohort study of patients diagnosed with ovarian, fallopian tube, and primary peritoneal cancer and receiving neoadjuvant chemotherapy from January 2009 to May 2014 at a single academic institution. The timing and number of venous thromboembolic events for the entire cohort were categorized as follows: presenting symptom, during neoadjuvant chemotherapy treatment, after debulking surgery, and during adjuvant chemotherapy. RESULTS: Of the 125 total patients with ovarian cancer undergoing neoadjuvant chemotherapy, 13 of 125 patients (10.4%, 95% confidence interval [CI] 6.1-17.2%) had a venous thromboembolism as a presenting symptom and were excluded from further analysis. Of the 112 total patients at risk, 30 (26.8%, 95% CI 19.3-35.9%) experienced a venous thromboembolism. Based on the phase of care, 13 (11.6%, 95% CI 6.8-19.1%) experienced a venous thromboembolism during neoadjuvant chemotherapy, six (5.4%, 95% CI 2.4-11.5%) developed a postoperative venous thromboembolism, and 11 (9.9%, 95% CI 5.5-17%) developed a venous thromboembolism during adjuvant chemotherapy. Two of the four patients with clear cell histology developed a venous thromboembolism in this cohort. CONCLUSION: Overall new diagnosis of venous thromboembolism was associated with one fourth of the patients undergoing neoadjuvant chemotherapy for ovarian cancer with nearly half of these diagnosed during chemotherapy cycles before interval debulking surgery. Efforts to reduce venous thromboembolism so far have largely focused on the postoperative period. Additional attention to venous thromboembolic prophylaxis during chemotherapy (neoadjuvant and adjuvant) in this patient population is warranted in an effort to decrease the rates of venous thromboembolism.

Effector T Cells Abrogate Stroma-Mediated Chemoresistance in Ovarian Cancer.

Effector T cells and fibroblasts are major components in the tumor microenvironment. The means through which these cellular interactions affect chemoresistance is unclear. Here, we show that fibroblasts diminish nuclear accumulation of platinum in ovarian cancer cells, resulting in resistance to platinum-based chemotherapy. We demonstrate that glutathione and cysteine released by fibroblasts contribute to this resistance. CD8(+) T cells abolish the resistance by altering glutathione and cystine metabolism in fibroblasts. CD8(+) T-cell-derived interferon (IFN)γ controls fibroblast glutathione and cysteine through upregulation of gamma-glutamyltransferases and transcriptional repression of system xc(-) cystine and glutamate antiporter via the JAK/STAT1 pathway. The presence of stromal fibroblasts and CD8(+) T cells is negatively and positively associated with ovarian cancer patient survival, respectively. Thus, our work uncovers a mode of action for effector T cells: they abrogate stromal-mediated chemoresistance. Capitalizing upon the interplay between chemotherapy and immunotherapy holds high potential for cancer treatment.

Resveratrol inhibits ovarian tumor growth in an in vivo mouse model.

BACKGROUND: Resveratrol inhibits the growth of ovarian carcinoma cells in vitro through the inhibition of glucose metabolism and the induction of both autophagy and apoptosis. In the current study, we investigated the metabolic and therapeutic effects of resveratrol in vivo. METHODS: A fluorescent xenograft mouse model of ovarian cancer was used. Mice were treated with cisplatin, resveratrol, or vehicle alone. Tumor burden was assessed using whole-body imaging. The effect of resveratrol on glucose uptake in vivo was determined using micro-positron emission tomography scanning. To determine whether resveratrol could inhibit tumor regrowth, tumor-bearing mice were treated with cisplatin followed by either daily resveratrol or vehicle. Autophagic response in resected tumors taken from mice treated with resveratrol was examined by transmission electron microscopy. Glycolysis and mitochondrial respiration in ovarian tumor cells after treatment with resveratrol was assessed. RESULTS: Mice treated with resveratrol and cisplatin were found to have a significantly reduced tumor burden compared with control animals (P<.001). Resveratrol-treated mice demonstrated a marked decrease in tumor uptake of glucose compared with controls. After treatment with cisplatin, "maintenance" resveratrol resulted in the suppression of tumor regrowth compared with mice receiving vehicle alone (P<.01). Tumors resected from mice treated with resveratrol exhibited autophagosomes consistent with the induction of autophagy. Treatment with resveratrol inhibited glycolytic response in ovarian tumor cells with high baseline glycolytic rates. CONCLUSIONS: Treatment with resveratrol inhibits glucose uptake and has a significant antineoplastic effect in a preclinical mouse model of ovarian cancer. Resveratrol treatment suppresses tumor regrowth after therapy with cisplatin, suggesting that this agent has the potential to prolong disease-free survival. Cancer 2016;122:722-729. © 2015 American Cancer Society.

A case of small cell carcinoma of the ovary hypercalcemic variant in a teenager.

► Young women with hypercalcemic type small cell ovarian cancer face a poor prognosis. ► Tumors respond to multi-agent chemotherapy, although rapid recurrence is typical. ► Using updated immunohistochemical staining patterns, this tumor can be identified.

Glucose deprivation activates AMPK and induces cell death through modulation of Akt in ovarian cancer cells.

OBJECTIVES: Upregulation of glycolysis has been demonstrated in multiple tumor types. Glucose deprivation results in diminished intracellular ATP; this is counteracted by AMPK activation during energy deficiency to restore ATP levels. We sought to determine whether glucose deprivation could induce cytotoxicity in ovarian cancer cells through activation of AMPK, and whether AMPK activators could mimic glucose deprivation induced cytotoxicity. METHODS: Sensitivity to 2DG induced cytotoxicity and glucose deprivation was determined in a panel of ovarian cancer cells. Cellular growth rate, rate of glucose uptake, and response to glucose deprivation were determined. Expression of Glut-1, HIF1-α, AMPK and Akt was determined by immunoblotting. RESULTS: Incubation of ovarian cancer cells with glucose-free media, 2-DG and AMPK activators resulted in cell death. The glycolytic phenotype of ovarian cancer cells was present in both normoxic and hypoxic conditions, and did not correlate with HIF1-α expression levels. Sensitivity to glucose deprivation was independent of growth rate, rate of glucose uptake, and appeared to be dependent upon constitutive activation of Akt. Glucose deprivation resulted in activation of AMPK and inhibition of Akt phosphorylation. Treatment with AMPK activators resulted in AMPK activation, Akt inhibition, and induced cell death in ovarian cancer cells. CONCLUSIONS: Ovarian cancer cells are glycolytic as compared to normal, untransformed cells, and are sensitive to glucose deprivation. Because ovarian cancer cells are dependent upon glucose for growth and survival, treatment with AMPK activators that mimic glucose deprivation may result in broad clinical benefits to ovarian cancer patients.

Intra-operative detection of nodal metastasis in early stage cervical cancer: a survey of the practice patterns of SGO members.

OBJECTIVES: To determine the practice patterns of members of Society of Gynecologic Oncologists (SGO) in different clinical situations involving the intra-operative detection of nodal metastasis in early stage cervical cancer. METHODS: A study questionnaire was mailed to the current members of SGO (n=874). Data were collected using an internet survey database. Frequency distributions were determined, and non parametric tests were performed. RESULTS: Thirty percent SGO members responded (n=274). Only 38.6% routinely performed an intra-operative frozen section evaluation of the lymph nodes. Of these; most (79%) did not abort the radical hysterectomy (RH) for an isolated microscopically positive pelvic lymph node. The likelihood of aborting RH for microscopic nodal involvement increased however with number of positive pelvic lymph nodes (21% with 1, 40% with 2-3, and 61% with >3 positive pelvic lymph nodes), involvement of para-aortic lymph nodes (61%), or bilaterally positive lymph nodes (54%). Similarly, a large number did not complete the RH due to gross involvement of pelvic (45%) or para-aortic lymph node/s (69%). Most (90%) completed the lymphadenectomy before aborting RH. When completing RH, the majority tailored its extent to perform a less radical resection. Variables significantly associated with the likelihood of completing RH in different clinical situations included: location of current practice (West), practice type (private), years in practice (>15 years), and number of cases seen per year (>10/month). CONCLUSION: Practice patterns of SGO members are considerably diverse, which is reflective of the conflicting evidence available in the literature. Well designed studies are required to determine the best overall approach.

Trichostatin A restores Apaf-1 function in chemoresistant ovarian cancer cells.

BACKGROUND: Chemoresistance is the major factor limiting long-term treatment success in patients with epithelial ovarian cancers. Most cytotoxic drugs kill cells through apoptosis; therefore, defective execution of apoptotic pathways results in a drug-resistant phenotype in many tumor types. METHODS: A panel of ovarian cancer cell lines was screened for expression and function of the apoptosome components Apaf-1 and caspase-9. Expression levels were analyzed by immunohistochemistry and immunoblotting; Apaf-1 function was determined by assessing the ability of endogenous Apaf-1 to cleave caspase-9 in the presence or absence of cytochrome c. The effect of the histone deacetylase inhibitor trichostatin A on Apaf-1 expression and function was evaluated. RESULTS: The authors report here that the resistance of ovarian cancer cells to the proapoptotic effects of chemotherapy is due in part to deficient Apaf-1 activity. Although Apaf-1 is expressed in most ovarian cancers, the functional activity is impaired, as Apaf-1 has a diminished ability to recruit and activate caspase-9. Treatment of ovarian cancer cells with trichostatin A results in restoration of Apaf-1 function independent of alterations in Apaf-1 expression. Furthermore, treating chemoresistant cells with sublethal doses of trichostatin A restores Apaf-1 function and sensitizes cells to cisplatin-induced apoptosis. CONCLUSIONS: Targeting intrinsic pathway defects for therapeutic intervention may result in sensitizing tumors to standard chemotherapy or triggering apoptosis in the absence of other apoptotic signals. The identification of drugs that can use Apaf-1 when it is present, yet can overcome its functional inactivation, may be an important clinical advance.

Validation of tumor size as staging variable in the revised International Federation of Gynecology and Obstetrics stage I leiomyosarcoma: a population-based study.

INTRODUCTION: Tumor size has been introduced as a staging variable in the 2008 International Federation of Gynecology and Obstetrics (FIGO) staging system for stage I leiomyosarcoma. In the prior 1988 FIGO staging system, leiomyosarcoma used the same staging criteria as endometrial cancer including cervical involvement. In this large population-based study, we validate the use of tumor size for purposes of risk stratification among stage I leiomyosarcoma patients. METHODS: Data were extracted from the Surveillance, Epidemiology, and End Results database between 1988 and 2005. Kaplan-Meier log rank and Cox proportional hazards models were used for survival analysis and to identify possible predictors for survival. RESULTS: The identified cohort included 819 women: 158 (19.3%), 2008 FIGO stage IA and 661 (80.7%), 2008 FIGO stage IB leiomyosarcoma. The 5-year overall survival rate was better in stage IA than in stage IB leiomyosarcoma (76.6% vs 48.4%, P < 0.001). Similarly, the 5-year overall survival rates were significantly different (P < 0.001) among women with different tumor size categories: 5 cm or smaller, 5.1 to 10 cm, and larger than 10 cm (76.6%, 52.9%, and 41.9%, respectively). The difference in 5-year overall survival rates between women with and without cervical involvement was significant (28.5% vs 55.3%, P = 0.014). Although age (P < 0.001), cervical involvement (P = 0.014), tumor grade (P < 0.001), tumor size (P < 0.001), performance of salpingo-oophorectomy (P = 0.001), and stage (P < 0.001) were all significant prognostic factors on univariate analysis, only age (P = 0.007), tumor size (P < 0.001), tumor grade (P < 0.001), and performance of salpingo-oophorectomy (P = 0.02) were significant predictors on multivariate analysis. Variables not found significant on univariate analysis (hence excluded from the Cox model) included lymphadenectomy, radiation, and race. CONCLUSIONS: The new staging system using tumor size is better for risk stratification in stage I leiomyosarcoma compared with the 1988 FIGO staging system of endometrial cancer.

Should radical hysterectomy be aborted on intraoperative detection of nodal tumor metastasis in early stage cervical cancer?

BACKGROUND/OBJECTIVES: In the United States, type 1 to 3 radical hysterectomy (RH) is used for the treatment of early stage (I-IIA) cervical cancer (ESCC). In the event that nodal metastasis is detected intraoperatively, completion of the procedure is controversial. Here, we present a review of the literature regarding clinical management of patients with ESCC with intraoperative diagnosis of nodal metastasis. MATERIALS AND METHODS: An electronic search of PubMed and OVID for the English-language literature published between 1980 and 2008 was performed. Studies regarding completion or abandonment of RH on intraoperative detection of nodal metastasis in ESCC were reviewed to evaluate the impact of either approach on overall survival, recurrence rate, and complication rate. RESULTS: This review was unable to document a difference in overall survival greater than 10% between the aborted and completed RH groups, and the difference was not statistically significant. No clear relationship was found between completion of RH and pelvic control or morbidity. Although urinary complications including bladder dysfunction and fistulae were unique to the completed RH group, radiation-related complications such as radiation cystitis, radiation proctitis, and bone necrosis were seen primarily in the aborted RH group. CONCLUSIONS: Given the lack of significant difference in survival, further studies are needed to evaluate the impact of completing versus aborting RH on pelvic control and morbidity to reach a definitive conclusion.

Inhibition of glycolysis enhances cisplatin-induced apoptosis in ovarian cancer cells.

OBJECTIVE: Up-regulation of glycolysis has been demonstrated in multiple tumor types and is believed to originate as an adaptive response to the selective pressure of the tumor microenvironment. We hypothesized that ovarian cancer cells are dependent on the glycolytic pathway for adenosine triphosphate generation and that this phenotype could be exploited for therapeutic intervention. STUDY DESIGN: Expression of glucose transporter 1 (Glut1), phosphorylated protein kinase B (pPKB/pAkt), and phosphorylated mammalian target of rapamycin (pmTOR) was assessed in ovarian carcinoma tumors and cell lines. Cells were incubated with 2-deoxyglucose and rapamycin; growth inhibition, viability, and mechanism of cell death were determined. RESULTS: Ovarian carcinoma cells overexpress Glut1, pAkt, and pmTOR compared with benign ovarian epithelial cells. 2-deoxyglucose and rapamycin markedly enhance apoptotic and nonapoptotic cell death in ovarian cancer cells. CONCLUSION: The glycolytic phenotype of ovarian cancer cells can be targeted for therapeutic intervention. Combined treatment modalities that target multiple cellular pathways hold promise for the treatment of chemoresistant ovarian cancer cells.

Expression of metabolically targeted biomarkers in endometrial carcinoma.

OBJECTIVES: The differential metabolic phenotype observed between malignant and non-transformed cells may constitute a biochemical basis for therapeutic intervention. Increased glucose uptake is one of the major metabolic changes found in malignant tumors, a process that is mediated by glucose transporters such as Glut1. Cellular growth can be regulated by mTOR in response to the nutrient milieu. In this study, we sought to determine if endometrial carcinoma cells express Glut1 and mTOR, and if inhibition of these factors is cytotoxic to endometrial carcinoma cells in vitro. METHODS: Expression of Glut1, pAkt, and pmTOR was assessed in tissue microarrays constructed from 42 type I and 34 type II endometrial tumors by immunohistochemistry, and in a panel of endometrial carcinoma cell lines. Representative endometrial carcinoma cells with wild type or mutant endogenous PTEN were treated with the glucose analog 2-deoxyglucose (2-DG) and rapamycin, an mTOR inhibitor or cisplatin. Inhibition of cell growth and mechanism of cell death was determined. RESULTS: Glut1, pAkt, and pmTOR were expressed strongly in both types I and II endometrial carcinoma. 2-DG and rapamycin induced apoptotic cell death in type I endometrial carcinoma cells, and profound growth inhibition and cytostasis in type II endometrial carcinoma cells. CONCLUSIONS: Glut1, pAkt, and pmTOR are overexpressed in endometrial carcinomas. Distinct alterations in the phosphatidylinositol 3'-kinase (PI3K) pathway upstream of mTOR, such as pAkt, may identify endometrial carcinoma patients who may benefit from adjuvant treatment with mTOR inhibitors and/or glucose analogs.

The HIV protease inhibitor saquinavir induces endoplasmic reticulum stress, autophagy, and apoptosis in ovarian cancer cells.

OBJECTIVE: HIV patients taking antiretroviral protease inhibitors have a lower incidence of infection-associated malignancies, leading to the hypothesis that these drugs have antineoplastic activity. Given the need for novel treatment approaches in ovarian cancer, we sought to determine whether the protease inhibitor saquinavir has antineoplastic activity in ovarian cancer cell lines, and to elucidate the mechanism through which this occurs. METHODS: A panel of ovarian cancer cell lines was treated with saquinavir. The effect of saquinavir on cell growth, viability, apoptotic and non-apoptotic cell death was determined. Stimulation of endoplasmic reticulum stress (ERS) response was assessed by immunoblotting for ERS regulators GRP78 and ATF6. Induction of autophagy was assessed using transmission electron microscopy (TEM), and confocal microscopy was performed to demonstrate changes in green fluorescent protein-labeled LC3 expression patterns. RESULTS: Saquinavir induced cell death in chemosensitive and chemoresistant ovarian cancer cells in a time- and dose-dependent manner. Saquinavir treatment resulted in caspase-dependent apoptosis and caspase-independent cell death characterized by induction of ERS and autophagy. Cellular morphology assessed by TEM revealed apoptotic, autophagic, and necrotic cell death. CONCLUSIONS: Saquinavir is an FDA-approved agent for the treatment of HIV, and our data suggest that it may also have clinical application in the treatment of ovarian cancer. Saquinavir induces endoplasmic reticulum stress, autophagy, and apoptosis in ovarian cancer cells. Given the challenges of chemoresistance in ovarian cancer, saquinavir may have particular benefit in the treatment of chemoresistant tumors that may respond to the induction of caspase-independent cell death by mechanisms such as autophagy.

Differential protein mapping of ovarian serous adenocarcinomas: identification of potential markers for distinct tumor stage.

Ovarian serous carcinomas (OSCs) comprise over half of all ovarian carcinomas and account for the majority of ovarian cancer-related deaths. We used a 2-dimensional liquid-based protein mapping strategy to characterize global protein expression patterns in 19 OSC tumor samples from 15 different patients to facilitate molecular classification of tumor stage. Protein expression profiles were produced, using pI-based separation in the first dimension and hydrophobicity-based separation in the second dimension, over a pH range of 4.0-7.0. Hierarchical clustering was applied to protein maps to indicate the tumor interrelationships. The 19 tumor samples could be classified into two different groups, one group associated with low stage (Stage 1) tumors and the other group associated with high stage (Stages 3/4) tumors. Proteins that were differentially expressed in different groups were selected for identification by LTQ-ESI-MS/MS. Fourteen of the selected proteins were overexpressed in the low stage tumors; 46 of the proteins were overexpressed in the high stage tumors. These proteins are known to play an important role in cellular functions such as glycolysis, protein biosynthesis, and cytoskeleton rearrangement and may serve as markers associated with different stages of OSCs. To further confirm the stage-dependent protein identifications, Lamin A/C and Vimentin expression in ovarian serous carcinomas was assessed by immunohistochemistry using ovarian tumor tissue microarrays for 66 samples.