KLF7: a new candidate biomarker and therapeutic target for high-grade serous ovarian cancer.

BACKGROUND: In spite of great progress in the surgical and clinical management, until now no significant improvement in overall survival of High-Grade Serous Ovarian Cancer (HGSOC) patients has been achieved. Important aspects for disease control remain unresolved, including unclear pathogenesis, high heterogeneity and relapse resistance after chemotherapy. Therefore, further research on molecular mechanisms involved in cancer progression are needed to find new targets for disease management. The Krüppel-like factors (KLFs) are a family of transcriptional regulators controlling several basic cellular processes, including proliferation, differentiation and migration. They have been shown to play a role in various cancer-relevant processes, in a context-dependent way. METHODS: To investigate a possible role of KLF family members as prognostic biomarkers, we carried out a bioinformatic meta-analysis of ovarian transcriptome datasets in different cohorts of late-stage HGSOC patients. In vitro cellular models of HGSOC were used for functional studies exploring the role of KLF7 in disease development and progression. Finally, molecular modelling and virtual screening were performed to identify putative KLF7 inhibitors. RESULTS: Bioinformatic analysis highlighted KLF7 as the most significant prognostic gene, among the 17 family members. Univariate and multivariate analyses identified KLF7 as an unfavourable prognostic marker for overall survival in late-stage TCGA-OV and GSE26712 HGSOC cohorts. Functional in vitro studies demonstrated that KLF7 can play a role as oncogene, driving tumour growth and dissemination. Mechanistic targets of KLF7 included genes involved in epithelial to mesenchymal transition, and in maintaining pluripotency and self-renewal characteristics of cancer stem cells. Finally, in silico analysis provided reliable information for drug-target interaction prediction. CONCLUSIONS: Results from the present study provide the first evidence for an oncogenic role of KLF7 in HGSOC, suggesting it as a promising prognostic marker and therapeutic target.

Clinical Value of lncRNA MEG3 in High-Grade Serous Ovarian Cancer.

Long non-coding RNAs (lncRNAs) are emerging as regulators in cancer development and progression, and aberrant lncRNA profiles have been reported in several cancers. Here, we evaluated the potential of using the maternally expressed gene 3 (MEG3) tissue level as a prognostic marker in high-grade serous ovarian cancer (HGSOC), the most common and deadliest gynecologic malignancy. To the aim of the study, we measured MEG3 transcript levels in 90 pre-treatment peritoneal biopsies. We also investigated MEG3 function in ovarian cancer biology. We found that high MEG3 expression was independently associated with better progression-free (p = 0.002) and overall survival (p = 0.01). In vitro and in vivo preclinical studies supported a role for MEG3 as a tumor suppressor in HGSOC, possibly through modulation of the phosphatase and tensin homologue (PTEN) network. Overall, results from this study demonstrated that decreased MEG3 is a hallmark for malignancy and tumor progression in HGSOC.

Identification and antitumor activity of a novel inhibitor of the NIMA-related kinase NEK6.

The NIMA (never in mitosis, gene A)-related kinase-6 (NEK6), which is implicated in cell cycle control and plays significant roles in tumorigenesis, is an attractive target for the development of novel anti-cancer drugs. Here we describe the discovery of a potent ATP site-directed inhibitor of NEK6 identified by virtual screening, adopting both structure- and ligand-based techniques. Using a homology-built model of NEK6 as well as the pharmacophoric features of known NEK6 inhibitors we identified novel binding scaffolds. Twenty-five compounds from the top ranking hits were subjected to in vitro kinase assays. The best compound, i.e. compound 8 ((5Z)-2-hydroxy-4-methyl-6-oxo-5-[(5-phenylfuran-2-yl)methylidene]-5,6-dihydropyridine-3-carbonitrile), was able to inhibit NEK6 with low micromolar IC50 value, also displaying antiproliferative activity against a panel of human cancer cell lines. Our results suggest that the identified inhibitor can be used as lead candidate for the development of novel anti-cancer agents, thus opening the possibility of new therapeutic strategies.

Uncovering the role of nuclear Lysyl oxidase (LOX) in advanced high grade serous ovarian cancer.

OBJECTIVE: Lysyl oxidase (LOX) is an enzyme that catalyzes the cross-linking of collagen and elastin in the extracellular matrix, thus controlling the tensile strength of tissues. Along with this primary function, there are evidences supporting a role for LOX in many critical biological functions, including gene expression regulation, cell growth, adhesion and migration. Accordingly, recent studies have supported a pivotal role for LOX in cancer progression and metastasis. The current study aimed at investigating the prognostic significance and the functional role of intracellular LOX in ovarian cancer. METHODS: To this end, we analyzed LOX expression by immunohistochemistry in archived tumor material from advanced high grade serous ovarian cancer (HGSOC) patients (n=70) and correlated data with clinicopathological parameters and with response to chemotherapy. In vitro experiments were also used to investigate the functional consequences of LOX expression on behavioral aspects of HGSOC cells. RESULTS: Our results showed that nuclear LOX expression is associated with unfavorable outcome in advanced HGSOC, being an independent prognostic factor for disease recurrence. Besides, high nuclear levels were seen to be associated with resistance to first-line chemotherapy. Through gene expression modulation experiments in HGSOC cell lines, we demonstrate that LOX positively regulates cell proliferation, migration and anchorage-independent growth. CONCLUSIONS: Collectively, our data suggest that LOX functions as a tumor promoter in HGSOC and positively regulates several aspects of the metastatic cascade.

GTP is an allosteric modulator of the interaction between the guanylate-binding protein 1 and the prosurvival kinase PIM1.

GBP1 and PIM1 are known to interact with a molar ratio 1:1. GBP1:PIM1 binding initiates a signaling pathway that induces resistance to common chemotherapeutics such as paclitaxel. Since GBP1 is a large GTPase which undergoes conformational changes in a nucleotide-dependent manner, we investigated the effect of GTP/GDP binding on GBP1:PIM1 interaction by using computational and biological studies. It resulted that only GTP decreases the formation of the GBP1:PIM1 complex through an allosteric mechanism, putting the bases for the identification of new compounds potentially able to revert resistance to paclitaxel.

Sox9 and Hif-2α regulate TUBB3 gene expression and affect ovarian cancer aggressiveness.

UNLABELLED: SOX9 [(sex determining region Y)-box9] gene has been implicated in the development and progression of different neoplasms. This study investigated the role of Sox9 in the expression of TUBB3 gene, a marker of aggressiveness in ovarian cancer (OC), encoding ßIII-tubulin protein. Gene expression was assessed by quantitative polymerase chain reaction (qPCR) in OC models. Using chromatin immunoprecipitation (ChIP) we found that Sox9 engages TUBB3 promoter at minus 980 base pairs from the transcriptional start site with transcriptional enhancing effects. Furthermore we found that Sox9 is a downstream target of Hif-2α, a transcription factor encoded by endothelial PAS domain protein-1 (EPAS1). Hypoxic microenvironment is a common feature of solid tumors associated with cancer aggressiveness. In the present work we found that knockdown of either SOX9 or EPAS1 abolished TUBB3 gene induction in hypoxia. This phenomenon was associated with a decrease in the number of cell colonies capable of growing in an anchorage-independent way. Using a nanofluidic genetic analyzer, the expression of SOX9, TUBB3 and EPAS1 was evaluated in 182 OC specimens. Double staining immunohistochemistry was employed to evaluate the expression and prognostic role of both Sox9 and ßIII-tubulin. Results obtained in cellular models matched the pattern of clinical specimens. We documented a direct correlation among the expression of EPAS1, SOX9 and TUBB3 at mRNA level. Patients displaying no expression for the three genes had the best outcome. A poor prognosis significant in multivariate analysis was visible in patients featuring high expression of ßIII-tubulin and nuclear Sox9. CONCLUSIONS: Sox9 allows the survival of OC cells upon hypoxic condition, through the activation of ßIII-tubulin expression and its aberrant activation in OC is prominent in patients with aggressive OC.

Gli family transcription factors are drivers of patupilone resistance in ovarian cancer.

Epothilones constitute a novel class of antitubulin agents that are active in patients who relapse after treatment with other chemotherapeutics. This study investigated the molecular mechanisms leading to the onset of epothilone-B (patupilone) resistance in ovarian cancer. Results demonstrated that the Gli family of transcription factors was overexpressed in resistant cells and that treatment with a specific Gli1 inhibitor (GANT58) made cells more susceptible to treatment, partially reversing drug resistance. We also demonstrated that Gli1 knockdown halted growth in resistant cells that were exposed to patupilone, confirming that Gli1 is capable of directly mediating epothilone-B resistance. Another observation from our research was that patupilone-resistant cells produced HGF and acquired characteristics of a mesenchymal phenotype. However, HGF silencing alone was not capable of converting the drug-resistant phenotype to a susceptible one, and in this case we demonstrated that Gli1 overexpression led to an increase in HGF, establishing a functional link between Gli1 and HGF. These results demonstrated that Gli1 played a key role in driving resistance to patupilone, suggesting that the combination of epothilones and Gli1-targeted agents could be exploited to improve outcomes in ovarian cancer patients resistant to standard treatments.

Class III ß-tubulin and the cytoskeletal gateway for drug resistance in ovarian cancer.

The Class III ß-tubulin isotype (ßIII-tubulin) is a predictive biomarker in ovarian cancer and other solid tumor malignancies. We discovered that ßIII-tubulin function is linked to two GTPases: guanylate-binding protein 1 (GBP1), which activates its function, and GNAI1, which inhibits it. This finding was demonstrated in a panel of ovarian cancer cells resistant to several chemotherapeutic agents. Using a protein microarray, we identified PIM1 as the downstream partner of GBP1, recruited into the cytoskeleton under hypoxic conditions. The clinical value of these observations was tested by performing an archive study of 98 ovarian cancer patients, which demonstrated that the ßIII-tubulin -/PIM1- cohort responded to treatment, exhibiting long overall survival (OS), while ßIII-tubulin +/PIM+ patients experienced poor outcomes and OS times similar to patients receiving palliation alone. ßIII-tubulin expression is commonly believed responsible for paclitaxel resistance due to its enhancement of the dynamic instability of microtubules, which counteracts the activity of taxanes. In contrast, our research reveals that ßIII-tubulin behaves as a gateway for prosurvival signals, such as PIM1, to move into the cytoskeleton. When cells are exposed to microenvironmental stressors, they activate this pathway by telling the cytoskeleton to incorporate PIM1 through GBP1 and ßIII-tubulin, which ultimately leads to drug resistance. This discovery reveals that ßIII-tubulin does not act alone but requires partners to play its role. The discovery of such protein:protein interactions underlying this prosurvival cascade makes feasible the development of therapeutic approaches using novel compounds that are capable of inhibiting the transmission of prosurvival signals into the cytoskeleton.

From plasma membrane to cytoskeleton: a novel function for semaphorin 6A.

Class III beta-tubulin (TUBB3) overexpression has been reported in ovary, lung, breast, and gastric cancer patients. Currently, no clinical drugs are available for a specific targeting of TUBB3, whereas the investigational drug IDN5390 specifically interacts with TUBB3. To gain insight into the pathways leading to TUBB3 up-regulation, we did a human genome microarray analysis in A2780 cells made resistant to IDN5390 to identify selected pathways specifically disrupted in resistant cells. Using this approach, we discovered that semaphorin 6A (SEMA6A) is down-regulated not only in IDN5390-resistant cells but also in cells made resistant to cisplatin, topotecan, and doxorubicin, whereas no changes were noticed in paclitaxel- and gemcitabine-resistant cells. Acute treatment with IDN5390 was able to down-regulate SEMA6A in cells unselected for drug resistance. TUBB3 expression was assessed in A2780 clones with stable overexpression of SEMA6A and in a panel of clones in which silencing of the protein was obtained. Quantitative PCR was then used to check the modulation of SEMA6A as well as to assess the expression of TUBB3. TUBB3 was increased (median value, 5.4) and reduced (median value, 0.47) in cells with overexpression and silencing of SEMA6A, respectively. Thus, the findings indicate a correlation between the expression of SEMA6A and TUBB3. Then, we found that a form of 83 kDa of SEMA6A is expressed in the cytoskeleton in association with beta-actin. These findings suggest for SEMA6A a novel function in the cytoskeleton and a role in modulating tubulin isotype composition and microtubule dynamics.