Light-induced Extracellular Vesicle Adsorption.

The role of extracellular vesicles (EVs) in human health and disease has garnered considerable attention over the past two decades. However, while several types of EVs are known to interact dynamically with the extracellular matrix and there is great potential value in producing high-fidelity EV micropatterns, there are currently no label-free, high-resolution, and tunable platform technologies with this capability. We introduce Light-induced Extracellular Vesicle Adsorption (LEVA) as a powerful solution to rapidly advance the study of matrix- and surface-bound EVs and other particles. The versatility of LEVA is demonstrated using commercial GFP-EV standards, EVs from glioblastoma bioreactors, and E. coli outer membrane vesicles (OMVs), with the resulting patterns used for single EV characterization, single cell migration on migrasome-mimetic trails, and OMV-mediated neutrophil swarming. LEVA will enable rapid advancements in the study of matrix- and surface-bound EVs and other particles, and should encourage researchers from many disciplines to create novel diagnostic, biomimetic, immunoengineering, and therapeutic screening assays.

Tissue factor as a novel diagnostic target for early detection of ovarian cancer using ultrasound microbubbles.

INTRODUCTION: Ovarian cancer (OC) is the deadliest gynecologic malignancy, with an overall 5-year survival rate of less than 30%. The existing paradigm for OC detection involves a serum marker, CA125, and ultrasound examination, neither of which is sufficiently specific for OC. This study addresses this deficiency through the use of a targeted ultrasound microbubble directed against tissue factor (TF). METHODS: TF expression was examined in both OC cell lines and patient-derived tumor samples via western blotting and IHC. In vivo microbubble ultrasound imaging was analyzed using high grade serous ovarian carcinoma orthotopic mouse models. RESULTS: While TF expression has previously been described on angiogenic, tumor-associated vascular endothelial cells (VECs) of several tumor types, this is first study to show TF expression on both murine and patient-derived ovarian tumor-associated VECs. Biotinylated anti-TF antibody was conjugated to streptavidin-coated microbubbles and in vitro binding assays were performed to assess the binding efficacy of these agents. TF-targeted microbubbles successfully bound to TF-expressing OC cells, as well as an in vitro model of angiogenic endothelium. In vivo, these microbubbles bound to the tumor-associated VECs of a clinically relevant orthotopic OC mouse model. CONCLUSION: Development of a TF-targeted microbubble capable of successfully detecting ovarian tumor neovasculature could have significant implications towards increasing the number of early-stage OC diagnoses. This preclinical study shows potential for translation to clinical use, which could ultimately help increase the number of early OC detections and decrease the mortality associated with this disease.

Targeting TMEM205 mediated drug resistance in ovarian clear cell carcinoma using oncolytic virus.

BACKGROUND: Ovarian clear cell carcinoma (OCCC) accounts for approximately 8-10% of epithelial ovarian cancers in the United States. Although it is rare, OCCC usually presents with treatment challenges and the overall prognosis is far worse than high grade serous ovarian cancer HGSOC. The objective of this study was to examine the therapeutic relevance of combining oncolytic virus with cisplatin for ovarian cancer clear cell carcinoma (OCCC). RESULTS: We identified that TMEM205, a recently discovered transmembrane protein, contributes to chemoresistance in OCCC cells via the exosomal pathway. Mechanistically, TMEM205 undergoes ligand-independent constitutive endocytosis and co-localizes with Rab11 to contribute to the late recycling endosomes in a clathrin-independent manner. Further, we observed that oncolytic virus (oHSV) pretreatment followed by treatment with cisplatin decreases TMEM205 expression and sensitizes cells to cisplatin in a synergistic manner in OCCC cells. TMEM205 interacts with glycoprotein-C of oHSV post-infection; both of these proteins undergo ubiquitination and ultimately get shuttled outside the cell via exosomes. Thus, we demonstrate the mechanotransduction pathway of TMEM205-mediated chemoresistance along with targeting this pathway using oHSV and cisplatin as a powerful therapeutic strategy for OCCC. oHSV combination with cisplatin inhibits OCCC tumor growth in vivo in immunodeficient and immunocompetent mice models. CONCLUSION: Our results suggest that the combination of oHSV and cisplatin in immunocompetent as well as immune deficient OCCC tumor bearing mice reduces overall tumor burden as well as metastatic disease thereby providing survival benefit. Additionally, the detection of TMEM205 in exosomal cargo early in OCCC development has potential to be exploited as a biomarker.

Aberrant expression of TMEM205 signaling promotes platinum resistance in ovarian cancer: An implication for the antitumor potential of DAP compound.

INTRODUCTION: TMEM205 is a novel transmembrane protein associated with platinum resistance (PR) in epithelial ovarian carcinoma (OC), however, the specific mechanisms associated with this resistance remain to be elucidated. METHODS: TMEM205 expression was evaluated in platinum-sensitive (PS) versus platinum resistant (PR) ovarian cancer cell lines and patient serum/tissues. Exosomal efflux of platinum was evaluated with inductively coupled plasma mass spectrometry (ICP-MS) after pre-treatment with small molecule inhibitors (L-2663/L-2797) of TMEM205 prior to treatment with platinum. Cytotoxicity of combination treatment was confirmed in vitro and in an in vivo model. RESULTS: TMEM205 expression was 10-20 fold higher in PR compared to PS ovarian cancer cell lines, serum samples, and tissues. Co-localization with CD1B was confirmed by in-situ proximity ligation assay suggesting that TMEM205 may mediate PR via the exosomal pathway. Exosomal secretion was significantly increased 5-10 fold in PR cell lines after treatment with carboplatin compared to PS cell lines. Pre-treatment with L-2663 prior to carboplatin resulted in significantly increased intracellular concentration of fluorescently-labeled cisplatin and decreased exosomal efflux of platinum. Decreased cell survival and tumor growth in vitro and in vivo was observed when PR cells were treated with a combination of L-2663 with carboplatin compared to carboplatin alone. CONCLUSION: TMEM205 appears to be involved in the development of PR in ovarian cancer through the exosomal efflux of platinum agents. This study provides pre-clinical evidence that TMEM205 could serve as a possible biomarker for PR as well as a therapeutic target in combination with platinum agents.

Oncolytic HSV Therapy Modulates Vesicular Trafficking Inducing Cisplatin Sensitivity and Antitumor Immunity.

PURPOSE: Here we investigated the impact of oncolytic herpes simplex virus (HSV) treatment on cisplatin sensitivity of platinum-resistant ovarian cancer, and the impact of the combination on immunotherapy. EXPERIMENTAL DESIGN: Therapeutic efficacy of the combination was assessed in platinum-resistant human and murine ovarian cancer peritoneal metastatic mouse models (n = 9-10/group). RNA sequencing along with flow cytometry of splenocytes from treated mice was employed to examine the effect of antitumor immune response (n = 3/group). Anti-PD-1 antibody was performed to evaluate impact on checkpoint inhibition in vivo. RESULTS: Gene Ontology pathway analysis uncovered disruption of cellular extracellular vesicle (EV)-related pathways in infected cells (FDR = 2.97E-57). Mechanistically, we identified reduced expression of transporters expressed on EV implicated in cisplatin efflux. The increased cisplatin retention led to increased cisplatin-DNA adducts, which resulted in micronuclei and the subsequent activation of cGAS-STING pathway with a significant activation of innate immune cells and translated to an increase in antitumor immunity and efficacy. In mice bearing platinum-resistant ovarian cancer, we also observed a feedback induction of PD-L1 on tumor cells, which sensitized combination-treated mice to anti-PD-1 immune checkpoint therapy. CONCLUSIONS: To our knowledge, this is the first report to show HSV-induced cisplatin retention in infected cells. The consequential increased damaged DNA was then expelled from cells as micronuclei which resulted in induction of inflammatory responses and education of antitumor immunity. The combination therapy also created an environment that sensitized tumors to immune checkpoint therapy.

HIF-transcribed p53 chaperones HIF-1α.

Chronic hypoxia is associated with a variety of physiological conditions such as rheumatoid arthritis, ischemia/reperfusion injury, stroke, diabetic vasculopathy, epilepsy and cancer. At the molecular level, hypoxia manifests its effects via activation of HIF-dependent transcription. On the other hand, an important transcription factor p53, which controls a myriad of biological functions, is rendered transcriptionally inactive under hypoxic conditions. p53 and HIF-1α are known to share a mysterious relationship and play an ambiguous role in the regulation of hypoxia-induced cellular changes. Here we demonstrate a novel pathway where HIF-1α transcriptionally upregulates both WT and MT p53 by binding to five response elements in p53 promoter. In hypoxic cells, this HIF-1α-induced p53 is transcriptionally inefficient but is abundantly available for protein-protein interactions. Further, both WT and MT p53 proteins bind and chaperone HIF-1α to stabilize its binding at its downstream DNA response elements. This p53-induced chaperoning of HIF-1α increases synthesis of HIF-regulated genes and thus the efficiency of hypoxia-induced molecular changes. This basic biology finding has important implications not only in the design of anti-cancer strategies but also for other physiological conditions where hypoxia results in disease manifestation.

A Microfluidic Chip Enables Isolation of Exosomes and Establishment of Their Protein Profiles and Associated Signaling Pathways in Ovarian Cancer.

Because of limits on specificity and purity to allow for in-depth protein profiling, a standardized method for exosome isolation has yet to be established. In this study, we describe a novel, in-house microfluidic-based device to isolate exosomes from culture media and patient samples. This technology overcomes contamination issues because sample separation is based on the expression of highly specific surface markers CD63 and EpCAM. Mass spectrometry revealed over 25 exosome proteins that are differentially expressed in high-grade serous ovarian cancer (HGSOC) cell lines compared with normal cells-ovarian surface epithelia cells and fallopian tube secretory epithelial cells (FTSEC). Top exosome proteins were identified on the basis of their fold change and statistical significance between groups. Ingenuity pathway analysis identified STAT3 and HGF as top regulator proteins. We further validated exosome proteins of interest (pSTAT3, HGF, and IL6) in HGSOC samples of origin-based cell lines (OVCAR-8, FTSEC) and in early-stage HGSOC patient serum exosome samples using LC/MS-MS and proximity extension assay. Our microfluidic device will allow us to make new discoveries for exosome-based biomarkers for the early detection of HGSOC and will contribute to the development of new targeted therapies based on signaling pathways that are unique to HGSOC, both of which could improve the outcome for women with HGSOC. SIGNIFICANCE: A unique platform utilizing a microfluidic device enables the discovery of new exosome-based biomarkers in ovarian cancer.

Inhibition of miR-328-3p Impairs Cancer Stem Cell Function and Prevents Metastasis in Ovarian Cancer.

Cancer stem cells (CSC) play a central role in cancer metastasis and development of drug resistance. miRNA are important in regulating CSC properties and are considered potential therapeutic targets. Here we report that miR-328-3p (miR-328) is significantly upregulated in ovarian CSC. High expression of miR-328 maintained CSC properties by directly targeting DNA damage binding protein 2, which has been shown previously to inhibit ovarian CSC. Reduced activity of ERK signaling in ovarian CSC, mainly due to a low level of reactive oxygen species, contributed to the enhanced expression of miR-328 and maintenance of CSC. Inhibition of miR-328 in mouse orthotopic ovarian xenografts impeded tumor growth and prevented tumor metastasis. In summary, our findings provide a novel mechanism underlying maintenance of the CSC population in ovarian cancer and suggest that targeted inhibition of miR-328 could be exploited for the eradication of CSC and aversion of tumor metastasis in ovarian cancer. SIGNIFICANCE: These findings present inhibition of miR-328 as a novel strategy for efficient elimination of CSC to prevent tumor metastasis and recurrence in patients with epithelial ovarian cancer.

Microfluidic affinity separation chip for selective capture and release of label-free ovarian cancer exosomes.

Exosomes are nanoscale vesicles found in many bodily fluids which play a significant role in cell-to-cell signaling and contain biomolecules indicative of their cells of origin. Recently, microfluidic devices have provided the ability to efficiently capture exosomes based on specific membrane biomarkers, but releasing the captured exosomes intact and label-free for downstream characterization and experimentation remains a challenge. We present a herringbone-grooved microfluidic device which is covalently functionalized with antibodies against general and cancer exosome membrane biomarkers (CD9 and EpCAM) to isolate exosomes from small volumes of high-grade serous ovarian cancer (HGSOC) serum. Following capture, intact exosomes are released label-free using a low pH buffer and immediately neutralized downstream to ensure their stability. Characterization of captured and released exosomes was performed using fluorescence microscopy, nanoparticle tracking analysis, flow-cytometry, and SEM. Our results demonstrate the successful isolation of intact and label-free exosomes, indicate that the amount of both total and EpCAM+ exosomes increases with HGSOC disease progression, and demonstrate the downstream internalization of isolated exosomes by OVCAR8 cells. This device and approach can be utilized for a nearly limitless range of downstream exosome analytical and experimental techniques, both on and off-chip.

Hypoxia-induced exosomes contribute to a more aggressive and chemoresistant ovarian cancer phenotype: a novel mechanism linking STAT3/Rab proteins.

Hypoxia-mediated tumor progression, metastasis, and drug resistance are major clinical challenges in ovarian cancer. Exosomes released in the hypoxic tumor microenvironment may contribute to these challenges by transferring signaling proteins between cancer cells and normal cells. We observed that ovarian cancer cells exposed to hypoxia significantly increased their exosome release by upregulating Rab27a, downregulating Rab7, LAMP1/2, NEU-1, and also by promoting a more secretory lysosomal phenotype. STAT3 knockdown in ovarian cancer cells reduced exosome release by altering the Rab family proteins Rab7 and Rab27a under hypoxic conditions. We also found that exosomes from patient-derived ascites ovarian cancer cell lines cultured under hypoxic conditions carried more potent oncogenic proteins-STAT3 and FAS that are capable of significantly increasing cell migration/invasion and chemo-resistance in vitro and tumor progression/metastasis in vivo. Hypoxic ovarian cancer cells derived exosomes (HEx) are proficient in re-programming the immortalized fallopian tube secretory epithelial cells (FT) to become pro-tumorigenic in mouse fallopian tubes. In addition, cisplatin efflux via exosomes was significantly increased in ovarian cancer cells under hypoxic conditions. Co-culture of HEx with tumor cells led to significantly decreased dsDNA damage and increased cell survival in response to cisplatin treatment. Blocking exosome release by known inhibitor Amiloride or STAT3 inhibitor and treating with cisplatin resulted in a significant increase in apoptosis, decreased colony formation, and proliferation. Our results demonstrate that HEx are more potent in augmenting metastasis/chemotherapy resistance in ovarian cancer and may serve as a novel mechanism for tumor metastasis, chemo-resistance, and a point of intervention for improving clinical outcomes.

The curcumin analog HO-3867 selectively kills cancer cells by converting mutant p53 protein to transcriptionally active wildtype p53.

p53 is an important tumor-suppressor protein that is mutated in more than 50% of cancers. Strategies for restoring normal p53 function are complicated by the oncogenic properties of mutant p53 and have not met with clinical success. To counteract mutant p53 activity, a variety of drugs with the potential to reconvert mutant p53 to an active wildtype form have been developed. However, these drugs are associated with various negative effects such as cellular toxicity, nonspecific binding to other proteins, and inability to induce a wildtype p53 response in cancer tissue. Here, we report on the effects of a curcumin analog, HO-3867, on p53 activity in cancer cells from different origins. We found that HO-3867 covalently binds to mutant p53, initiates a wildtype p53-like anticancer genetic response, is exclusively cytotoxic toward cancer cells, and exhibits high anticancer efficacy in tumor models. In conclusion, HO-3867 is a p53 mutant-reactivating drug with high clinical anticancer potential.

STAT3/PIAS3 Levels Serve as "Early Signature" Genes in the Development of High-Grade Serous Carcinoma from the Fallopian Tube.

The initial molecular events that lead to malignant transformation of the fimbria of the fallopian tube (FT) through high-grade serous ovarian carcinoma (HGSC) remain poorly understood. In this study, we report that increased expression of signal transducer and activator of transcription 3 (pSTAT3 Tyr705) and suppression or loss of protein inhibitor of activated STAT3 (PIAS3) in FT likely drive HGSC. We evaluated human tissues-benign normal FT, tubal-peritoneal junction (TPJ), p53 signature FT tissue, tubal intraepithelial lesion in transition (TILT), serous tubal intraepithelial carcinoma (STIC) without ovarian cancer, and HGSC for expression of STAT3/PIAS3 (compared with their known TP53 signature) and their target proliferation genes. We observed constitutive activation of STAT3 and low levels or loss of PIAS3 in the TPJ, p53 signature, TILT, and STIC through advanced stage IV (HGSC) tissues. Elevated expression of pSTAT3 Tyr705 and decreased levels of PIAS3 appeared as early as TPJ and the trend continued until very advanced stage HGSC (compared with high PIAS3 and low pSTAT3 expression in normal benign FT). Exogenous expression of STAT3 in FT cells mediated translocation of pSTAT3 and c-Myc into the nucleus. In vivo experiments demonstrated that overexpression of STAT3 in FT secretory epithelial cells promoted tumor progression and metastasis, mimicking the clinical disease observed in patients with HGSC. Thus, we conclude that the STAT3 pathway plays a role in the development and progression of HGSC from its earliest premalignant states.Significance: Concomitant gain of pSTAT3 Tyr705 and loss of PIAS3 appear critical for initiation and development of high-grade serous carcinoma. Cancer Res; 78(7); 1739-50. ©2018 AACR.

MAX Mutations in Endometrial Cancer: Clinicopathologic Associations and Recurrent MAX p.His28Arg Functional Characterization.

Background: Genomic studies have revealed that multiple genes are mutated at varying frequency in endometrial cancer (EC); however, the relevance of many of these mutations is poorly understood. An EC-specific recurrent mutation in the MAX transcription factor p.His28Arg was recently discovered. We sought to assess the functional consequences of this hotspot mutation and determine its association with cancer-relevant phenotypes. Methods: MAX was sequenced in 509 endometrioid ECs, and associations between mutation status and clinicopathologic features were assessed. EC cell lines stably expressing MAXH28R were established and used for functional experiments. DNA binding was examined using electrophoretic mobility shift assays and chromatin immunoprecipitation. Transcriptional profiling was performed with microarrays. Murine flank (six to 11 mice per group) and intraperitoneal tumor models were used for in vivo studies. Vascularity of xenografts was assessed by MECA-32 immunohistochemistry. The paracrine pro-angiogenic nature of MAXH28R-expressing EC cells was tested using microfluidic HUVEC sprouting assays and VEGFA enzyme-linked immunosorbent assays. All statistical tests were two-sided. Results: Twenty-two of 509 tumors harbored mutations in MAX, including 12 tumors with the p.His28Arg mutation. Patients with a MAX mutation had statistically significantly reduced recurrence-free survival (hazard ratio = 4.00, 95% confidence interval = 1.15 to 13.91, P = .03). MAXH28R increased affinity for canonical E-box sequences, and MAXH28R-expressing EC cells dramatically altered transcriptional profiles. MAXH28R-derived xenografts statistically significantly increased vascular area compared with MAXWT and empty vector tumors (P = .003 and P = .008, respectively). MAXH28R-expressing EC cells secreted nearly double the levels of VEGFA compared with MAXWT cells (P = .03, .005, and .005 at 24, 48, and 72 hours, respectively), and conditioned media from MAXH28R cells increased sprouting when applied to HUVECs. Conclusion: These data highlight the importance of MAX mutations in EC and point to increased vascularity as one mechanism contributing to clinical aggressiveness of EC.

Targeting STAT3 by HO3867 induces apoptosis in ovarian clear cell carcinoma.

Advanced ovarian clear cell carcinoma (OCCC) carries a very poor prognosis in large part secondary to the extremely high rate of resistance to standard platinum and taxane chemotherapy. Signal transducer and activator of transcription 3(STAT3) expression and activation has been shown to regulate tumor progression in various human cancers, though has not been well studied in OCCC. Preliminary work in our lab has demonstrated constitutive activation of STAT3 (pSTAT3Tyr705 or pSTAT3727) in OCCC cell lines as well as human OCCC tumor tissue samples. Significantly, pSTAT3 is expressed in the absence of other forms of activated STAT (pSTAT1, 2, 6). Therefore, this work was planned to investigate the role of STAT3 and examine the efficacy of a novel anti-cancer compound -HO-3867, which is an inhibitor of STAT3, using known OCCC cell lines. Results demonstrate that treatment with HO-3867 decreased expression of pSTAT3 Tyr705 as well pSTAT3 Ser727, while total STAT3 remained constant. STAT3 overexpression increased the migration capability in OVTOKO cells in vitro and led to an increased tumor size when injected in vivo. The inhibitory effect of HO-3867 on cell proliferation and cell survival was accompanied by increased apoptosis, within 24 h post treatment. Treatment with HO-3867 resulted in a decrease in Bcl-2 and increase of cleavage of caspase 3, caspase 7, and PARP, confirming induction of apoptosis after treatment with HO-3867. In addition, HO-3867 significantly inhibited formation of human umbilical vein endothelial cells capillary-like structures and invasion at both 5 and 10 µM concentrations. STAT3 expression plays an important role in the spread of OCCC in vitro as well as in vivo. Thus, we can exploit the STAT3 pathway for targeted drug therapy. Inhibition of pSTAT3 using HO-3867in OCCC cell lines appears to be a promising therapy. This is of utmost importance given the poor response of OCCC to standard chemotherapy regimens.

High Glucose-Mediated STAT3 Activation in Endometrial Cancer Is Inhibited by Metformin: Therapeutic Implications for Endometrial Cancer.

OBJECTIVES: STAT3 is over-expressed in endometrial cancer, and diabetes is a risk factor for the development of type 1 endometrial cancer. We therefore investigated whether glucose concentrations influence STAT3 expression in type 1 endometrial cancer, and whether such STAT3 expression might be inhibited by metformin. METHODS: In Ishikawa (grade 1) endometrial cancer cells subjected to media with low, normal, or high concentrations of glucose, expression of STAT3 and its target proteins was evaluated by real-time quantitative PCR (qPCR). Ishikawa cells were treated with metformin and assessed with cell proliferation, survival, migration, and ubiquitin assays, as well as Western blot and qPCR. Expression of apoptosis proteins was evaluated with Western blot in Ishikawa cells transfected with a STAT3 overexpression plasmid and treated with metformin. A xenograft tumor model was used for studying the in vivo efficacy of metformin. RESULTS: Expression of STAT3 and its target proteins was increased in Ishikawa cells cultured in high glucose media. In vitro, metformin inhibited cell proliferation, survival and migration but induced apoptosis. Metformin reduced expression levels of pSTAT3 ser727, total STAT3, and its associated cell survival and anti-apoptotic proteins. Additionally, metformin treatment was associated with increased degradation of pSTAT3 ser727. No change in apoptotic protein expression was noticed with STAT3 overexpression in Ishikawa cells. In vivo, metformin treatment led to a decrease in tumor weight as well as reductions of STAT3, pSTAT3 ser727, its target proteins. CONCLUSIONS: These results suggest that STAT3 expression in type 1 endometrial cancer is stimulated by a high glucose environment and inhibited by metformin.

HDAC10 as a potential therapeutic target in ovarian cancer.

OBJECTIVE: We analyzed histone deacetylase 10 (HDAC10) for function in the context of the DNA damage response in BRCA1-null ovarian cancer cells as well as evaluated the potential of general HDAC inhibitors in primary ovarian carcinoma cells. HDAC10 had previously been shown to be highly stimulatory to the process of homology directed repair in HeLa cells, and in this study we investigated whether HDAC10 could impact in vitro the response to anticancer therapies. We hypothesized that the loss of HDAC10 would sensitize cells to platinum therapy. METHODS: We combined informatics analysis of large DNA sequencing datasets from ovarian cancer tumors with tissue culture based assays of primary and established cell lines to test for sensitivity to platinum therapy if HDAC10 activity was inhibited or depleted. RESULTS: Using The Cancer Genome Atlas (TCGA) dataset, we found that deep deletions in HDAC10 occurred in 5-10% of ovarian cancer tumors. From the TCGA data we found that low HDAC10 mRNA levels correlated with platinum sensitivity of the tumors. Cell proliferation and DNA damage assays in a BRCA1-null ovarian carcinoma cell line demonstrated reduced DNA repair capacity and sensitization of platinum therapy. Similarly, primary ovarian carcinoma cells demonstrated a sensitization to platinum therapies when treated with HDAC inhibitors. CONCLUSIONS: From the results of this study, we suggest that the inhibition of HDAC10 may potentiate the effects of platinum therapies in ovarian tumors.

Elevated STAT3 expression in ovarian cancer ascites promotes invasion and metastasis: a potential therapeutic target.

Although activation of the STAT3 pathway has been associated with tumor progression in a wide variety of cancer types (including ovarian cancer), the precise mechanism of invasion and metastasis due to STAT3 are not fully delineated in ovarian cancer. We found that pSTAT3 Tyr705 is constitutively activated in patient ascites and ascites-derived ovarian cancer cells (ADOCCs), and the range of STAT3 expression could be very high to low. In vivo transplantation of ADOCCs with high pSTAT3 expression into the ovarian bursa of mice resulted in a large primary tumor and widespread peritoneal metastases. In contrast, ADOCCs with low STAT3 expression or ADOCCs with STAT3 expression knockdown, led to reduced tumor growth and an absence of metastases in vivo. Cytokines derived from the ADOCC culture medium activate the interleukin (IL)-6/STAT pathway in the STAT3 knockout (KO) cells, compensating for the absence of inherent STAT3 in the cells. Treatment with HO-3867 (a novel STAT3 inhibitor at 100 p.p.m. in an orthotopic murine model) significantly suppressed ovarian tumor growth, angiogenesis and metastasis by targeting STAT3 and its downstream proteins. HO-3867 was found to have cytotoxic effects in ex vivo cultures of freshly collected human ovarian cancers, including those resistant to platinum-based chemotherapy. Our results show that STAT3 is necessary for ovarian tumor progression/metastasis and highlight the potential for targeting STAT3 by HO-3867 as a therapeutic strategy for ovarian cancer.

Autophagy Induction Results in Enhanced Anoikis Resistance in Models of Peritoneal Disease.

Peritoneal carcinomatosis and peritoneal sarcomatosis is a potential complication of nearly all solid tumors and results in profoundly increased morbidity and mortality. Despite the ubiquity of peritoneal carcinomatosis/peritoneal sarcomatosis, there are no clinically relevant targeted therapies for either its treatment or prevention. To identify potential therapies, we developed in vitro models of peritoneal carcinomatosis/peritoneal sarcomatosis using tumor cell lines and patient-derived spheroids (PDS) that recapitulate anoikis resistance and spheroid proliferation across multiple cancer types. Epithelial- and mesenchymal-derived cancer cell lines (YOU, PANC1, HEYA8, CHLA10, and TC71) were used to generate spheroids and establish growth characteristics. Differential gene expression analyses of these spheroids to matched adherent cells revealed a consensus spheroid signature. This spheroid signature discriminates primary tumor specimens from tumor cells found in ascites of ovarian cancer patients and in our PDS models. Key in this gene expression signature is BNIP3 and BNIP3L, known regulators of autophagy and apoptosis. Elevated BNIP3 mRNA expression is associated with poor survival in ovarian cancer patients and elevated BNIP3 protein, as measured by IHC, and is also associated with higher grade tumors and shorter survival. Pharmacologic induction of autophagy with rapamycin significantly increased spheroid formation and survival while decreasing the induction of apoptosis. In contrast, the autophagy inhibitor hydroxychloroquine abrogated spheroid formation with a clear increase in apoptosis. Modulation of BNIP3 and the critical autophagy gene Beclin-1 (BECN1) also caused a significant decrease in spheroid formation. Combined, these data demonstrate how modulation of BNIP3-related autophagy, in PDS and in vitro spheroid models, alters the survival and morphology of spheroids. IMPLICATIONS: Development of BNIP3/BNIP3L-targeting agents or autophagy-targeting agents may reduce morbidity and mortality associated with peritoneal carcinomatosis and sarcomatosis. Mol Cancer Res; 15(1); 26-34. ©2016 AACR.

Anticancer potential of diarylidenyl piperidone derivatives, HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer.

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.