Epithelial-mesenchymal-transition (EMT) in response to TGFβ contributes to normal development, wound healing and tumor progression. The present study provides evidence for a critical role of β5-integrin in the TGFβ-induced EMT and the tumorigenic potential of carcinoma cells. We show that the αvβ-integrin subunits are upregulated during the TGFβ-induced EMT and this response requires Smad transcription factors. Depletion of αv-integrin by siRNA blocked the EMT response whereas knock-down of β1-integrin had no effect. Importantly, depletion of β5-integrin blocked the TGFβ-induced EMT impairing adhesion to cell-matrix and integrin signaling, but did not change expression of E-cadherin and TGFβ-target genes. Accordingly, the EMT process and integrin signaling were blocked by cRGD peptide interfering with cell-matrix adhesion or by inhibition of focal adhesion kinase, indicating the importance of β5-integrin-mediated adhesions in EMT. Finally, depletion of β5-integrin significantly reduced invasiveness of breast carcinoma cells. Thus, the β5-integrin adhesions contribute to the TGFβ-induced EMT and the tumorigenic potential of carcinoma cells.

No abstract

TGF-beta and Ras regulate epithelial-mesenchymal transition (EMT), a process that contributes to tumor invasion and metastasis. The interaction of these pathways in EMT is still poorly understood. Here, we show that TGF-beta induces EMT but limits cell invasion whereas hyperactivated Ras (H-RasV12) does not cause EMT but enhances cell invasion, alleviating the inhibitory effect of TGF-beta. TGF-beta disrupts cell junctions and induces tropomyosin-mediated actin fibers and matrix adhesion. Smad transcription factors mediate both steps of the TGF-beta-induced EMT whereas RasV12 inhibits the second step by blocking the induction of tropomyosins (TPM1) and reducing cell-matrix adhesion and integrin signaling. RasV12 prevents binding of Smads to the TPM1 promoter by forcing CRM1-dependent nuclear export of Smad4. Soft agar and animal studies demonstrate that RasV12 confers the metastatic potential in epithelial cells, whereas tropomyosin suppresses tumor growth and metastases. Thus, TGF-beta-induced EMT is not sufficient for the acquisition of the invasive potential and activated Ras alters this TGF-beta response, conferring the tumorigenic and invasive potential.

The prognosis of advanced/recurrent cervical cancer patients remains poor. We analyzed 54 fresh-frozen and 15 primary cervical cancer cell lines, along with matched-normal DNA, by whole-exome sequencing (WES), most of which harboring Human-Papillomavirus-type-16/18. We found recurrent somatic missense mutations in 22 genes (including PIK3CA, ERBB2, and GNAS) and a widespread APOBEC cytidine deaminase mutagenesis pattern (TCW motif) in both adenocarcinoma (ACC) and squamous cell carcinomas (SCCs). Somatic copy number variants (CNVs) identified 12 copy number gains and 40 losses, occurring more often than expected by chance, with the most frequent events in pathways similar to those found from analysis of single nucleotide variants (SNVs), including the ERBB2/PI3K/AKT/mTOR, apoptosis, chromatin remodeling, and cell cycle. To validate specific SNVs as targets, we took advantage of primary cervical tumor cell lines and xenografts to preclinically evaluate the activity of pan-HER (afatinib and neratinib) and PIK3CA (copanlisib) inhibitors, alone and in combination, against tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway (71%). Tumors harboring ERBB2 (5.8%) domain mutations were significantly more sensitive to single agents afatinib or neratinib when compared to wild-type tumors in preclinical in vitro and in vivo models (P = 0.001). In contrast, pan-HER and PIK3CA inhibitors demonstrated limited in vitro activity and were only transiently effective in controlling in vivo growth of PIK3CA-mutated cervical cancer xenografts. Importantly, combinations of copanlisib and neratinib were highly synergistic, inducing long-lasting regression of tumors harboring alterations in the ERBB2/PI3K/AKT/mTOR pathway. These findings define the genetic landscape of cervical cancer, suggesting that a large subset of cervical tumors might benefit from existing ERBB2/PIK3CA/AKT/mTOR-targeted drugs.

The loss of anti-proliferative responsiveness in prostate cancer cell lines toward ligands for vitamin D receptor, retinoic acid receptors/retinoid X receptors and peroxisome proliferator activated receptor (PPAR)alpha/gamma may entail underlying epigenetic events, as ligand insensitivity reflects significantly altered messenger RNA expression of corepressors and histone-modifying enzymes. Expression patterns were dependent on phases of the cell cycle and associated with repressed basal gene expression of vitamin D receptor and PPARalpha/gamma target genes, for example CDKN1A [encodes p21((waf1/cip1))]. Elevated nuclear corepressor 1 (NCOR1) and nuclear corepressor 2/silencing mediator of retinoic acid and thyroid hormone receptor protein levels were detected in prostate cancer cell lines compared with non-malignant counterparts. Knockdown of the corepressor NCOR1 significantly elevated basal expression of a cohort of target genes, including CDKN1A. Both chemical [histone deacetylases inhibitor (HDACi)] and NCOR1 knockdown targeting enhanced anti-proliferative sensitivity toward PPARalpha/gamma ligands in prostate cancer cell lines. Pursuing PPARalpha/gamma signaling, microarray approaches were undertaken to identify pathways and genes regulated uniquely by a combination of PPARalpha/gamma activation and HDAC inhibition. Again, HDACi and knockdown approaches demonstrated that elevated NCOR1 expression and activity distorted PPARalpha/gamma gene targets centered on, for example cell cycle control, including CDKN1A and TGFBRAP1. Quantitative real time polymerase chain reaction validation and chromatin immunoprecipitation assays both confirmed that elevated NCOR1 disrupted the ability of PPARalpha/gamma to regulate key target genes (CDKN1A and TGFBRAP1). Interrogation of these relationships in prostate cancer samples using principal component and partial correlation analyses established significant interdependent relationships between NCOR1-PPARalpha/gamma and representative target genes, independently of androgen receptor expression. Therefore, we conclude that elevated NCOR1 distorts the actions of PPARalpha/gamma selectively and generates a potential epigenetic lesion with diagnostic and prognostic significance.

The four-marker panel, CA125, HE4, E-CAD, and IL-6, shows potential in detecting serous ovarian cancer at earlier stages. Additional validation studies using the biomarker combination in ovarian cancer patients are warranted.

NA was well tolerated in all patients. A strong suppression of disease activity was observed in the majority of patients during the follow-up.

Ovarian cancer remains the most lethal gynecologic malignancy. We analyzed the mutational landscape of 64 primary, 41 metastatic, and 17 recurrent fresh-frozen tumors from 77 patients along with matched normal DNA, by whole-exome sequencing (WES). We also sequenced 13 pairs of synchronous bilateral ovarian cancer (SBOC) to evaluate the evolutionary history. Lastly, to search for therapeutic targets, we evaluated the activity of the Bromodomain and Extra-Terminal motif (BET) inhibitor GS-626510 on primary tumors and xenografts harboring c-MYC amplifications. In line with previous studies, the large majority of germline and somatic mutations were found in BRCA1/2 (21%) and TP53 (86%) genes, respectively. Among mutations in known cancer driver genes, 77% were transmitted from primary tumors to metastatic tumors, and 80% from primary to recurrent tumors, indicating that driver mutations are commonly retained during ovarian cancer evolution. Importantly, the number, mutation spectra, and signatures in matched primary–metastatic tumors were extremely similar, suggesting transcoelomic metastases as an early dissemination process using preexisting metastatic ability rather than an evolution model. Similarly, comparison of SBOC showed extensive sharing of somatic mutations, unequivocally indicating a common ancestry in all cases. Among the 17 patients with matched tumors, four patients gained PIK3CA amplifications and two patients gained c-MYC amplifications in the recurrent tumors, with no loss of amplification or gain of deletions. Primary cell lines and xenografts derived from chemotherapy-resistant tumors demonstrated sensitivity to JQ1 and GS-626510 (P = 0.01), suggesting that oral BET inhibitors represent a class of personalized therapeutics in patients harboring recurrent/chemotherapy-resistant disease.