MELK expression in ovarian cancer correlates with poor outcome and its inhibition by OTSSP167 abrogates proliferation and viability of ovarian cancer cells.

OBJECTIVE: Maternal embryonic leucine-zipper kinase (MELK) shows oncogenic properties in basal-like breast cancer, a cancer subtype sharing common molecular features with high-grade serous ovarian cancer. We examined the potential of MELK as a molecular and pharmacological target for treatment of epithelial ovarian cancer (EOC). METHODS/MATERIALS: Bioinformatic analysis was performed on nine OC transcriptomic data sets totaling 1241 patients. Effects of MELK depletion by shRNA or inhibition by OTSSP167 in cell lines were assessed by colony formation and MTT (proliferation) assays, Western blotting (apoptosis), and flow cytometry (cell cycle analysis). RESULTS: Elevated MELK expression was correlated with histological grading (n=6 data sets, p<0.05) and progression-free survival (HR 5.73, p<0.01) in OC patients and elevated MELK expression in other cancers with disease-free survival (n=3495, HR 1.071, p<0.001). Inhibition or depletion of MELK reduced cell proliferation and anchorage-dependent and -independent growth in various OC cell lines through a G2/M cell cycle arrest, eventually resulting in apoptosis. OTSSP167 retained its cytotoxicity in Cisplatin- and Paclitaxel-resistant IGROV1 and TYK-nu OC cells and sensitized OVCAR8 cells to Carboplatin but not Paclitaxel. CONCLUSION: MELK inhibition by OTSSP167 may thus present a strategy to treat patients with aggressive, progressive, and recurrent ovarian cancer.

Glucosylceramide synthase inhibitors differentially affect expression of glycosphingolipids.

Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration.

Alterations in the mitochondrial responses to PENAO as a mechanism of resistance in ovarian cancer cells.

OBJECTIVE: The purpose of this study was to test PENAO, a promising new organoarsenical that is in phase 1 testing in patients with solid tumours, on a range of ovarian cancer cell lines with different histotypes, and to understand the molecular basis of drug resistance exhibited by the endometrioid ovarian cancer cell line, SKOV-3. METHODS: Proliferation arrest and cell death induced by PENAO in serous (OVCAR-3), endometrioid (SKOV-3, TOV112D), clear cell (TOV21G) and mucinous (EFO27) ovarian cancer cells in culture, and anti-tumour efficacy in a murine model of SKOV-3 and OVCAR-3 tumours, were measured. Cells were analysed for cell cycle arrest, cell death mechanisms, reactive oxygen species production, mitochondrial depolarisation, oxygen consumption and acid production. RESULTS: PENAO demonstrated promising anti-proliferative activity on the most common (serous, endometrioid) as well as on rare (clear cell, mucinous) subtypes of ovarian cancer cell lines. No cross-resistance with platinum-based drugs was evident. Endometrioid SKOV-3 cells were, however, shown to be resistant to PENAO in vitro and in a xenograft mouse model. This resistance was due to an ability to cope with PENAO-induced oxidative stress, notably through heme oxygenase-1 induction, and a shift in metabolism towards glycolysis. The adaptive glycolytic shift in SKOV-3 was targeted using a mTORC1 inhibitor in combination with PENAO. This strategy was successful with the two drugs acting synergistically to inhibit cell proliferation and to induce cell death via apoptosis and autophagy. CONCLUSION: Mitochondria/mTOR dual-targeting therapy may constitute a new approach for the treatment of recurrent/resistant forms of epithelial ovarian cancer.

Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells.

BACKGROUND: The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated. RESULTS: RT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2'-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells. CONCLUSIONS: GBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.

Loss of ARID1A/BAF250a-expression in endometriosis: a biomarker for risk of carcinogenic transformation?

Mutations of the tumor-suppressor gene ARID1A result in the loss of protein expression of the BRG-associated factor 250a (BAF250a), a large subunit of transcription-regulating Human SWI/SNF complexes, which have an important role in the control of cell proliferation and tumor suppression. ARID1A mutations are particularly frequent in endometriosis-associated ovarian clear cell and endometrioid carcinomas, and were recently described as a possible key mechanism and early step in the transformation of endometriosis into cancer. Here, we examined the immunohistochemical expression pattern of BAF250a in a tissue microarray including 74 endometriosis and 30 endometrium samples. Ovarian cancer samples (n=136) served as a control. Epithelial BAF250a expression was assessable in 90/104 (87%) and stromal BAF250a expression in 95/104 (91%) of the endometriosis, and endometrium cases due to lack of adequate tissue in some spots. Complete lack of BAF250a expression was observed in three endometriomas (n=3/20, 15%) and one deep-infiltrating endometriosis sample (n=1/22, 5%), but in none of the peritoneal endometriosis (n=0/16) and eutopic endometrium samples (n=0/30). A comparison of the mean immunoreactivity scores revealed a significantly lower expression rate of BAF250a in endometriomas compared with normal endometrium (P<0.0005), as well as peritoneal (P=0.003) and deep-infiltrating endometriosis (P=0.02). Our data demonstrates that a complete loss of BAF250a expression is observable in some endometriotic lesions, especially in endometriomas. In addition, we report that a partial loss of BAF250a expression is occurring in the form of cell clusters indicating a clonal loss of BAF250a expression in these cells. The loss of expression of the tumor-suppressor protein BAF250a in some endometriomas possibly indicates a risk of malignant transformation in these cases, which could be of importance in the determination of individual treatment strategies. However, its role and value as a prognostic parameter in endometriosis needs to be further studied.

Expression of the G protein-coupled estrogen receptor (GPER) in endometriosis: a tissue microarray study.

BACKGROUND: The G protein-coupled estrogen receptor (GPER) is thought to be involved in non-genomic estrogen responses as well as processes such as cell proliferation and migration. In this study, we analyzed GPER expression patterns from endometriosis samples and normal endometrial tissue samples and compared these expression profiles to those of the classical sex hormone receptors. METHODS: A tissue microarray, which included 74 samples from different types of endometriosis (27 ovarian, 19 peritoneal and 28 deep-infiltrating) and 30 samples from normal endometrial tissue, was used to compare the expression levels of the GPER, estrogen receptor (ER)-alpha, ER-beta and progesterone receptor (PR). The immunoreactive score (IRS) was calculated separately for epithelium and stroma as the product of the staining intensity and the percentage of positive cells. The expression levels of the hormonal receptors were dichotomized into low (IRS < 6) and high (IRS > = 6) expression groups. RESULTS: The mean epithelial IRS (+/- standard deviation, range) of cytoplasmic GPER expression was 1.2 (+/- 1.7, 0-4) in normal endometrium and 5.1 (+/- 3.5, 0-12) in endometriosis (p < 0.001), of nuclear GPER 6.4 (+/- 2.6, 0-12) and 6.8 (+/- 2.9, 2-12; p = 0.71), of ER-alpha 10.6 (+/- 2.4, 3-12) and 9.8 (+/- 3.0, 2-12; p = 0.26), of ER-beta 2.4 (+/- 2.2; 0-8) and 5.6 (+/- 2.6; 0-10; p < 0.001), and of PR 11.5 (+/- 1.7; 3-12) and 8.1 (+/- 4.5; 0-12; p < 0.001), respectively. The mean stromal IRS of nuclear GPER expression was 7.7 (+/- 3.0; 2-12) in endometrium and 10.8 (+/- 1.7; 6-12) in endometriosis (p < 0.001), of ER-alpha 8.7 (+/- 3.1; 2-12) and 10.6 (+/- 2.4; 2-12; p = 0.001), of ER-beta 1.8 (+/- 2.0; 0-8) and 5.4 (+/- 2.5; 0-10; p < 0.001), and of PR 11.7 (+/- 0.9; 8-12) and 10.9 (+/- 2.0; 3-12; p = 0.044), respectively. Cytoplasmic GPER expression was not detectable in the stroma of endometrium and endometriosis. The observed frequency of high epithelial cytoplasmic GPER expression levels was 50% (n = 30/60) in the endometriosis and none (0/30) in the normal endometrium samples (p < 0.001). High epithelial cytoplasmic GPER expression levels were more frequent in endometriomas (14/20, 70%; p = 0.01), as compared to peritoneal (9/18, 50%) or deep-infiltrating endometriotic lesions (7/22, 31.8%). The frequency of high stromal nuclear GPER expression levels was 100% (n = 74/74) in endometriosis and 76.7% (n = 23/30) in normal endometrium (p < 0.001). The frequency of high epithelial nuclear GPER expression levels did not differ between endometriosis and normal endometrium. CONCLUSIONS: The present data indicate a unique GPER expression pattern in endometriosis, especially in endometriomas as compared to the normal endometrium. The overexpression of GPER in endometriotic lesions suggests a potential role for GPER in the hormonal regulation of endometriosis, which should be taken into consideration for future hormonal treatment strategies.

Exposure to escalating olaparib does not induce acquired resistance to PARPi and to other chemotherapeutic compounds in ovarian cancer cell lines.

Poly (ADP­ribose) polymerase (PARP)­inhibitors (PARPi) such as olaparib and niraparib are currently used as a treatment option for BRCA­deficient tumors and also show efficacy in platinum­sensitive tumors. However, resistance to PARPi occurs in numerous patients and in particular acquired PARPi resistance presents a major obstacle in the treatment of these tumors. In the present study, it was investigated whether stepwise exposure of ovarian cancer cells to escalating concentrations of olaparib produced subcells with acquired resistance to PARPi and/or acquired cross­resistance to platinum compounds, paclitaxel, and doxorubicin. To this aim, the sensitivity of fourteen ovarian cancer cell lines, including nine with TP53­mutations and five carrying BRCA­mutations, to olaparib and niraparib was determined and a subset of seven cell lines was selected to investigate the potential of olaparib to produce resistance. It was identified that escalating olaparib did neither produce subcells with acquired PARPi­resistance nor did it produce acquired cross­resistance to platinum compounds, doxorubicin, and paclitaxel. This finding was independent of the cells' TP53 and BRCA mutation status. CRISPR­Cas9 mediated deletion of PARP1 did not affect sensitivity to PARPi, platinum compounds, doxorubicin, and paclitaxel. In addition, olaparib sensitivity correlated with niraparib sensitivity, but BRCA­mutated cells were not more sensitive to PARPi. Moreover, PARPi sensitivity associated with cross­sensitivity not only to platinum compounds but also to anthracylines, paclitaxel, and inhibitors of histone deacetylases. These in vitro data indicated that olaparib exposure is unlikely to produce an acquired resistance phenotype and that PARPi­sensitive ovarian cancer cells are also cross­sensitive to non­platinum and even to compounds not directly interacting with the DNA.

Overlapping gene dependencies for PARP inhibitors and carboplatin response identified by functional CRISPR-Cas9 screening in ovarian cancer.

PARP inhibitors (PARPi) have revolutionized the therapeutic landscape of epithelial ovarian cancer (EOC) treatment with outstanding benefits in regard to progression-free survival, especially in patients either carrying BRCA1/2 mutations or harboring defects in the homologous recombination repair system. Yet, it remains uncertain which PARPi to apply and how to predict responders when platinum sensitivity is unknown. To shed light on the predictive power of genes previously suggested to be associated with PARPi response, we systematically reviewed the literature and identified 79 publications investigating a total of 93 genes. The top candidate genes were further tested using a comprehensive CRISPR-Cas9 mutagenesis screening in combination with olaparib treatment. Therefore, we generated six constitutive Cas9+ EOC cell lines and profiled 33 genes in a CRISPR-Cas9 cell competition assay using non-essential (AAVS1) and essential (RPA3 and PCNA) genes for cell fitness as negative and positive controls, respectively. We identified only ATM, MUS81, NBN, BRCA2, and RAD51B as predictive markers for olaparib response. As the major survival benefit of PARPi treatment was reported in platinum-sensitive tumors, we next assessed nine top candidate genes in combination with three PARPi and carboplatin. Interestingly, we observed similar dropout rates in a gene and compound independent manner, supporting the strong correlation of cancer cell response to compounds that rely on DNA repair for their effectiveness. In addition, we report on CDK12 as a common vulnerability for EOC cell survival and proliferation without altering the olaparib response, highlighting its potential as a therapeutic target in EOC.

MyCTC chip: microfluidic-based drug screen with patient-derived tumour cells from liquid biopsies.

Cancer patients with advanced disease are characterized by intrinsic challenges in predicting drug response patterns, often leading to ineffective treatment. Current clinical practice for treatment decision-making is commonly based on primary or secondary tumour biopsies, yet when disease progression accelerates, tissue biopsies are not performed on a regular basis. It is in this context that liquid biopsies may offer a unique window to uncover key vulnerabilities, providing valuable information about previously underappreciated treatment opportunities. Here, we present MyCTC chip, a novel microfluidic device enabling the isolation, culture and drug susceptibility testing of cancer cells derived from liquid biopsies. Cancer cell capture is achieved through a label-free, antigen-agnostic enrichment method, and it is followed by cultivation in dedicated conditions, allowing on-chip expansion of captured cells. Upon growth, cancer cells are then transferred to drug screen chambers located within the same device, where multiple compounds can be tested simultaneously. We demonstrate MyCTC chip performance by means of spike-in experiments with patient-derived breast circulating tumour cells, enabling >95% capture rates, as well as prospective processing of blood from breast cancer patients and ascites fluid from patients with ovarian, tubal and endometrial cancer, where sensitivity to specific chemotherapeutic agents was identified. Together, we provide evidence that MyCTC chip may be used to identify personalized drug response patterns in patients with advanced metastatic disease and with limited treatment opportunities.

Glycosphingolipids are mediators of cancer plasticity through independent signaling pathways.

The molecular repertoire promoting cancer cell plasticity is not fully elucidated. Here, we propose that glycosphingolipids (GSLs), specifically the globo and ganglio series, correlate and promote the transition between epithelial and mesenchymal cells. The epithelial character of ovarian cancer remains stable throughout disease progression, and spatial glycosphingolipidomics reveals elevated globosides in the tumor compartment compared with the ganglioside-rich stroma. CRISPR-Cas9 knockin mediated truncation of endogenous E-cadherin induces epithelial-to-mesenchymal transition (EMT) and decreases globosides. The transcriptomics analysis identifies the ganglioside-synthesizing enzyme ST8SIA1 to be consistently elevated in mesenchymal-like samples, predicting poor outcome. Subsequent deletion of ST8SIA1 induces epithelial cell features through mTORS2448 phosphorylation, whereas loss of globosides in ΔA4GALT cells, resulting in EMT, is accompanied by increased ERKY202/T204 and AKTS124. The GSL composition dynamics corroborate cancer cell plasticity, and further evidence suggests that mesenchymal cells are maintained through ganglioside-dependent, calcium-mediated mechanisms.

Expression pattern of class I histone deacetylases in vulvar intraepithelial neoplasia and vulvar cancer: a tissue microarray study.

BACKGROUND: Epigenetic regulation is an important mechanism leading to cancer initiation and promotion. Histone acetylation by histone deacetylases (HDACs) represents an important part of it. The development of HDAC inhibitors has identified the utility of HDACs as a therapeutic target. Little is known about the epigenetic regulation of vulvar intraepithelial neoplasia (VIN) and vulvar squamous cell cancer (VSCC). In this study, the expression of class I HDACs (HDAC 1, 2 and 3) was compared in a series of VIN and VSCC tissues. METHODS: A tissue micro array (TMA) with specimens from 106 patients with high-grade VIN and 59 patients with vulvar cancer was constructed. The expression of HDACs 1, 2 and 3 were analyzed with immunohistochemistry (IHC). The nuclear expression pattern was evaluated in terms of intensity and percentage of stained nuclei and was compared between vulvar preinvasive lesions and vulvar cancer. RESULTS: HDAC 2 expression was significantly higher in VIN than in VSCC (p < 0.001, Fisher's test). Also, 88.7% (n = 94/106) of VIN samples and only 54.5% (n = 31/57) of VSCC samples were scored at the maximum level. Conversely, HDAC 3 expression was significantly higher in VSCC (93%, 53/57) compared to VIN (73.6%, 78/106, p = 0.003), whereas only a small difference in the expression of HDAC 1 was found between these two entities of vulvar neoplasia. CONCLUSIONS: These results suggest that epigenetic regulation plays a considerable role in the transformation of VIN to invasive vulvar neoplasia.

Cordycepin (3'-deoxyadenosine), an inhibitor of mRNA polyadenylation, suppresses proliferation and activates apoptosis in human epithelial endometriotic cells in vitro.

BACKGROUND/AIMS: Endometriosis is a benign but chronic disorder associated with pelvic pain and infertility. Enhanced proliferation and reduced apoptosis susceptibility are characteristics of endometriosis. Cordycepin is a poly(A) polymerase inhibitor. It induces shortening of poly(A) tails, leading to destabilization of mRNAs and finally to proliferation inhibition and cell death in normal and tumor cells. The potential of cordycepin to block proliferation and survival of 11z human immortalized epithelial endometriotic cells was determined. METHODS: 11z cell cultures were treated with cordycepin. Cordycepin-induced inhibition of proliferation and alterations in protein expression and protein phosphorylation were determined by the methyl thiazolyl tetrazolium assay and immunoblot analysis, respectively. RESULTS: Cordycepin induced the rapid and significant upregulation of the cell cycle progression inhibitor p21 and the downregulation of the cell cycle progression promoter cyclin D(1), finally leading to the inhibition of the proliferation of 11z human epithelial endometriotic cells. Cordycepin reduced the phosphorylation of the p38 mitogen-activated protein kinase and the retinoblastoma protein. It also activated caspase-dependent, intrinsic apoptosis, as documented by the proteolytic cleavage of the caspase-9, caspase-3 and the poly(ADP ribose) polymerase 1 precursor. CONCLUSION: The mRNA polyadenylation inhibitor cordycepin inhibits proliferation and survival of endometriotic cells.

Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis.

The extracellular matrix (ECM) plays critical roles in tumor progression and metastasis. However, the contribution of ECM proteins to early metastatic onset in the peritoneal cavity remains unexplored. Here, we suggest a new route of metastasis through the interaction of integrin alpha 2 (ITGA2) with collagens enriched in the tumor coinciding with poor outcome in patients with ovarian cancer. Using multiple gene-edited cell lines and patient-derived samples, we demonstrate that ITGA2 triggers cancer cell adhesion to collagen, promotes cell migration, anoikis resistance, mesothelial clearance, and peritoneal metastasis in vitro and in vivo. Mechanistically, phosphoproteomics identify an ITGA2-dependent phosphorylation of focal adhesion kinase and mitogen-activated protein kinase pathway leading to enhanced oncogenic properties. Consequently, specific inhibition of ITGA2-mediated cancer cell-collagen interaction or targeting focal adhesion signaling may present an opportunity for therapeutic intervention of metastatic spread in ovarian cancer.

MLH1 protects from resistance acquisition by the histone deacetylase inhibitor trichostatin A in colon tumor cells.

The antineoplastic activity of HDAC inhibitors is an unquestionable property of these compounds, but recent studies in tumor cells have revealed the potential of HDAC inhibitors (e.g., suberoylanilide hydroxamic acid SAHA, valproic acid VPA) to cause acquisition of HDAC inhibitor resistance. We report that trichostatin A (TSA), an HDAC inhibitor structurally related to SAHA, causes the acquisition of multidrug resistance transporter-independent and irreversible 3-fold resistance to TSA in MLH1-deficient (absent MLH1 protein expression) but not in MLH1-proficient (expressing MLH1 protein) HCT116 colon tumor cells. This MLH1-deficient subline selected for TSA resistance by stepwise exposures to increasing TSA concentrations exhibited failure in the accumulation of acetylated histones, in p21 induction, and in apoptosis activation. These are cellular responses normally seen in tumor cells treated with HDAC inhibitors. Whereas the absence of acetyl-histone accumulation did not correlate with altered HDAC activity, the absence of apoptosis correlated with reduced expression of (pro-apoptotic) Bax. This TSA-resistant subline was cross-resistant to SAHA and VPA but not to 'classic' non-HDAC inhibitor-type anticancer agents such as docetaxel and doxorubicin. These herein presented results expand on a previous study reporting HDAC inhibitor resistance acquisition by SAHA which was independent of the MLH1 expression status. Taken together, the present study identifies TSA, besides SAHA and VPA, as another potential causative of HDAC inhibitor resistance acquisition specifically in MLH1-deficient HCT116 colon tumor cells, and it reveals a possible function of MLH1 protein in protecting colon tumor cells from resistance acquisition by TSA.

Acquired vorinostat resistance shows partial cross-resistance to 'second-generation' HDAC inhibitors and correlates with loss of histone acetylation and apoptosis but not with altered HDAC and HAT activities.

Histone deacetylase (HDAC) inhibitors such as vorinostat (suberoylanilide hydroxamic acid), valproic acid, romidepsin (FK-228), and LBH589 comprise a relatively new class of potent anticancer agents. This study provides evidence for the potential of vorinostat to cause acquisition of multidrug resistance protein-independent resistance in HCT116 colon tumor cells. This acquired resistance is moderate (two-fold to three-fold), is nonreversible, and correlates with the loss of responses typically seen with HDAC inhibitors, that is the loss of acetylation of the histones H2A, H2B, H3, and H4, the loss of the G2/M checkpoint activation, and the loss of caspase 3-dependent and caspase 7-dependent apoptosis. This acquired resistance also associates with cross-resistance to the hydroxamate-class (LBH589 and JNJ26481585) and to the aliphatic acid-class (valproic acid) HDAC inhibitors but not to the benzamide-class (MGCD0103) and the cyclic peptide-class (romidepsin) HDAC inhibitors. The acquired HDAC inhibitor resistance described hereis not a result of altered HDAC and histone acetyltransferase activities and differs from that previously reported for romidepsin.

Outcome in serous ovarian cancer is not associated with LATS expression.

BACKGROUND: Large tumor suppressor (LATS) proteins are putative tumor suppressors and poorly expressed associated with poor outcome in many cancers. A recent immunohistochemistry study showed that LATS protein expression correlated with poor outcome in serous ovarian cancer. MATERIALS AND METHODS: We analyzed LATS expression in various ovarian cancer transcriptomic data sets and immunohistochemically assessed LATS protein expression in a Swiss ovarian tumor cohort. Results were compared to clinicopathological characteristics and outcome. We also compared LATS protein expression in serous ovarian cancer cell lines to their EMT status (Western blotting) and drug sensitivity (MTT assay). RESULTS: The analysis of 15 different transcriptomic data sets showed that LATS2 was associated with poorer outcome, while LATS1 was irrelevant (HR = 1.19 and HR = 1.00, respectively). The TCGA-RNASeqV2 data set showed that low LATS1 and LATS2 were associated with better survival in serous ovarian carcinoma. Despite heterogeneity among the different data sets, LATS expression is not an indicator of survival in serous ovarian cancer and LATS2 expression may even be tumorigenic. LATS expression was neither associated with survival nor with the stage and grade in the Swiss cohort. It was low in cystadenoma, intermediate in carcinoma, and high in borderline tumors and was higher in serous than mucinous ovarian carcinoma. LATS protein expression extent was comparable in epithelial-, intermediate-, and mesenchymal-type ovarian cancer cells and was not associated with drug sensitivity. CONCLUSION: These results are largely incompatible with a tumor-suppressive function of LATS in ovarian cancer, and LATS protein level is also not an indicator for drug sensitivity and EMT status of ovarian cancer cells.

Diaphragmatic smears are not of additional benefit in the detection of peritoneal spread in gynecological cancers.

Peritoneal biopsies (PB) and peritoneal washing (PW) are routine measures in abdominal staging of gynecological malignancies and are used particularly for the assessment of occult microscopic tumor spread to the peritoneal surface including the diaphragm. Cytological diaphragmatic smears (DS) have been suggested as a supplemental tool; however, they are not routinely taken and their usefulness is still unclear. The present study retrospectively evaluated whether DS provide an additional benefit over PB and PW for the detection of peritoneal malignancies in patients with gynecological cancer. The data from patients who underwent laparotomy for suspected gynecological cancer and had DS and either PB, PW or ascites were reviewed. Sensitivity and specificity, and the number upstaged patients were determined. A total of 43 patients were excluded due to benign diagnosis (those with negative DS or PW) and 2 out of the remaining had 2 carcinomas simultaneously. Among these 41 malignancies, DS were positive in 12, PW in 18 and PB in 19 cases. No case was DS-positive while negative for both PB and PW. Four cases were missed when only PB and 5 when only PW was performed. Notably, no case of peritoneal disease was identified solely on positive DS, indicating that all 23 positive cases (presence of occult peritoneal disease in 56.1%) were identified by PB and PW together (100% sensitivity; 62% specificity). In addition, none of the cases was upstaged solely on positive DS results. Taken together, these data demonstrated that DS do not present an additional benefit to PW and PB in the detection of peritoneal gynecological disease.

ABO blood groups as a prognostic factor for recurrence in ovarian and vulvar cancer.

The relationship between ABO blood groups (BG) and risk of incidence in cancers including gynecological cancers has been widely studied, showing increased incidence risk for BG A patients. As available data are inconsistent we investigated whether BG and their anti-glycan antibodies (anti-A and anti-B) have prognostic values in gynecological cancers. We retrospectively evaluated 974 patients with gynecological cancers in three cancer centers (Switzerland and Australia) between 1974 and 2014 regarding the relationships between clinico-pathological findings and the BG. Time to disease recurrence was significantly influenced by BG in patients with ovarian (n = 282) and vulvar (n = 67) cancer. BG O or B patients showed a significantly increased risk for ovarian cancer relapse compared to A, 59% and 82%, respectively (p = 0.045; HR O vs A = 1.59 (CI 1.01-2.51) and (p = 0.036; HR A vs B = 0.55 (CI 0.32-0.96). Median time to relapse for advanced stage (n = 126) ovarian cancer patients was 18.2 months for BG O and 32.2 for A (p = 0.031; HR O vs A = 2.07 (CI 1.07-4.02)). BG also significantly influenced relapse-free survival in patients with vulvar cancer (p = 0.002), with BG O tending to have increased relapse risk compared to A (p = 0.089). Blood groups hence associate with recurrence in ovarian and vulvar cancer: women with BG O seem to have a lower ovarian cancer incidence, however are more likely to relapse earlier. The significance of the BG status as a prognostic value is evident and may be helpful to oncologists in prognosticating disease outcome and selecting the appropriate therapy.

Transition of Mesenchymal and Epithelial Cancer Cells Depends on α1-4 Galactosyltransferase-Mediated Glycosphingolipids.

The reversible transitions of cancer cells between epithelial and mesenchymal states comprise cellular and molecular processes essential for local tumor growth and respective dissemination. We report here that globoside glycosphingolipid (GSL) glycosyltransferase-encoding genes are elevated in epithelial cells and correlate with characteristic EMT signatures predictive of disease outcome. Depletion of globosides through CRISPR-Cas9-mediated deletion of the key enzyme A4GALT induces EMT, enhances chemoresistance, and increased CD24low/CD44high cells. The cholera toxin-induced mesenchymal-to-epithelial transition occurred only in cells with functional A4GALT. Cells undergoing EMT lost E-cadherin expression through epigenetic silencing at the promoter region of CDH1 However, in ΔA4GALT cells, demethylation was able to rescue E-cadherin-mediated cell-cell adhesion only in the presence of exogenous A4GALT. Overall, our data suggest another class of biomolecules vital for epithelial cancer cells and for maintaining cell integrity and function.Significance: This study highlights the essential role of glycosphingolipids in the maintenance of epithelial cancer cell properties. Cancer Res; 78(11); 2952-65. ©2018 AACR.

The histone deacetylase inhibitors suberoylanilide hydroxamic (Vorinostat) and valproic acid induce irreversible and MDR1-independent resistance in human colon cancer cells.

Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA, Vorinostat), valproic acid (VPA), and FK228 are members of a relatively novel class of small molecular weight chemicals that have high antineoplastic activity. They cause growth inhibition and apoptosis specifically in tumor cells, and they act also as chemo- and radio-sensitizers. In the present study, the potential of SAHA and VPA to induce resistance was studied. To that aim HDAC inhibitor-resistant sublines were generated by stepwise exposure of colon tumor cells to increasing concentrations of these compounds. Clonogenic data demonstrated that the SAHA- and VPA-induced sublines were 2-fold resistant to these compounds. This resistance was non-reversible, as it was maintained even when the sublines were cultured in the absence of SAHA or VPA. The SAHA- and VPA-induced resistant sublines were also stably cross-resistant to VPA and SAHA, respectively, but retained sensitivity against non-HDAC inhibitor-type anticancer agents. The SAHA-induced resistance correlated with loss of the G2/M checkpoint but it was not accompanied by reduced induction of the endogenous cell cycle inhibitors p21 and p27. Furthermore, SAHA-induced resistance was not due to reduced apoptosis, and it was neither dependent on MDR expression nor was it due to increased expression of HDAC1 and HDAC3. Taken together, these data demonstrate the potential of SAHA and VPA to induce resistance. This resistance was not dependent on MDR expression, did not involve MMR, and seemed to underlie a mechanism that differs from that underlying the previously observed FK228-induced resistance. The finding that SAHA and VPA induce only modest resistance despite continuous treatment and that the resistance is MDR-independent suggests a preference for these two drugs over FK228 for use in combination treatment with classic anticancer agents.