DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

DIRAS3 regulates the autophagosome initiation complex in dormant ovarian cancer cells

DIRAS3 is an imprinted tumor suppressor gene that is downregulated in 60% of human ovarian cancers. Re-expression of DIRAS3 at physiological levels inhibits proliferation, decreases motility, induces autophagy, and regulates tumor dormancy. Functional inhibition of autophagy with choroquine in dormant xenografts that express DIRAS3 significantly delays tumor regrowth after DIRAS3 levels are reduced, suggesting that autophagy sustains dormant ovarian cancer cells. This study documents a newly discovered role for DIRAS3 in forming the autophagosome initiation complex (AIC) that contains BECN1, PIK3C3, PIK3R4, ATG14, and DIRAS3. Participation of BECN1 in the AIC is inhibited by binding of BECN1 homodimers to BCL2. DIRAS3 binds BECN1, disrupting BECN1 homodimers and displacing BCL2. Binding of DIRAS3 to BECN1 increases the association of BECN1 with PIK3C3 and ATG14, facilitating AIC activation. Amino acid starvation of cells induces DIRAS3 expression, reduces BECN1-BCL2 interaction and promotes autophagy, whereas DIRAS3 depletion blocks amino acid starvation-induced autophagy. In primary ovarian cancers, punctate expression of DIRAS3, BECN1, and the autophagic biomarker MAP1LC3 are highly correlated (P < 0.0001), underlining the clinical relevance of these mechanistic studies. Punctate expression of DIRAS3 and MAP1LC3 was detected in only 21–23% of primary ovarian cancers but in 81–84% of tumor nodules found on the peritoneal surface at second-look operations following primary chemotherapy. This reflects a 4-fold increase (P < 0.0001) in autophagy between primary disease and post-treatment recurrence. We suggest that DIRAS3 not only regulates the AIC, but induces autophagy in dormant, nutrient-deprived ovarian cancer cells that remain after conventional chemotherapy, facilitating their survival.     

Dormant Tumors Awaken by a Short-Term Angiogenic Burst: The Spike Hypothesis

Tumor dormancy, a complex and still poorly understood phenomenon observed both in experimental models and in patients, has been associated with insufficient angiogenic capacity. A defined event, termed "angiogenic switch" and characterized by an imbalance between pro- and anti-angiogenic factors, often marks interruption of the dormant state, thus triggering invasive tumor growth. In our current view, sustained angiogenesis is considered essential in promoting this transition. Recently, we demonstrated that co-administration of proliferation-arrested Kaposi's sarcoma cells or recombinant angiogenic factors interrupts dormancy of poorly angiogenic leukemia cells by providing a brief angiogenic burst. These findings indicate that even a transient angiogenic switch can prime progressive tumor growth and suggest that tumor angiogenesis is a process requiring a higher amount of angiogenic factors for its induction than maintenance. Here we discuss the implications of these observations on our view of tumor angiogenesis and on the therapeutic potential of angiogenesis inhibitors.     

Knockdown of KDM1A suppresses tumour migration and invasion by epigenetically regulating the TIMP1/MMP9 pathway in papillary thyroid cancer

Epigenetic dysregulation plays an important role in cancer. Histone demethylation is a well‐known mechanism of epigenetic regulation that promotes or inhibits tumourigenesis in various malignant tumours. However, the pathogenic role of histone demethylation modifiers in papillary thyroid cancer (PTC), which has a high incidence of early lymphatic metastasis, is largely unknown. Here, we detected the expression of common histone demethylation modifiers and found that the histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) demethylase KDM1A (or lysine demethylase 1A) is frequently overexpressed in PTC tissues and cell lines. High KDM1A expression correlated positively with age <55 years and lymph node metastasis in patients with PTC. Moreover, KDM1A was required for PTC cell migration and invasion. KDM1A knockdown inhibited the migration and invasive abilities of PTC cells both in vitro and in vivo. We also identified tissue inhibitor of metalloproteinase 1 (TIMP1) as a key KDM1A target gene. KDM1A activated matrix metalloproteinase 9 (MMP9) through epigenetic repression of TIMP1 expression by demethylating H3K4me2 at the TIMP1 promoter region. Rescue experiments clarified these findings. Altogether, we have uncovered a new mechanism of KDM1A repression of TIMP1 in PTC and suggest that KDM1A may be a promising therapeutic target in PTC.     

Model of Tumor Dormancy/Recurrence after Short-Term Chemotherapy

Although many tumors regress in response to neoadjuvant chemotherapy, residual tumor cells are detected in most cancer patients post-treatment. These residual tumor cells are thought to remain dormant for years before resuming growth, resulting in tumor recurrence. Considering that recurrent tumors are most often responsible for patient mortality, there exists an urgent need to study signaling pathways that drive tumor dormancy/recurrence. We have developed an in vitro model of tumor dormancy/recurrence. Short-term exposure of tumor cells (breast or prostate) to chemotherapy at clinically relevant doses enriches for a dormant tumor cell population. Several days after removing chemotherapy, dormant tumor cells regain proliferative ability and establish colonies, resembling tumor recurrence. Tumor cells from “recurrent” colonies exhibit increased chemotherapy resistance, similar to the therapy resistance of recurrent tumors in cancer patients. Previous studies using long-term chemotherapy selection models identified acquired mutations that drive tumor resistance. In contrast, our short term chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that can resume growth after drug removal. Studying unique signaling pathways in dormant tumor cells enriched by short-term chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.     

PARP inhibitor Veliparib (ABT-888) enhances the anti-angiogenic potentiality of Curcumin through deregulation of NECTIN-4 in oral cancer: Role of nitric oxide (NO)

Concurrent use of DNA damaging agents with PARP inhibitors contribute to the effectiveness of the anticancer therapy. But there is a dearth of reports on the antiangiogenic effects of PARP inhibitors and the suppression of angiogenesis by this drug combination is not yet reported. For the successful development of cancer therapeutics, anti-cancer drugs ought to have anti-angiogenic potentiality along with their DNA damaging abilities. In this current piece of work, we investigated the in vitro and in ovo anti-angiogenic effect of Curcumin and Veliparib (a PARP inhibitor) in oral cancer. Recent evidences suggest an involvement of the NECTIN-4 in cancer angiogenesis and the exact molecular pathway of this involvement remains to be delineated. We observed that the soluble NECTIN-4 secreted from H357 oral cancer cells enhanced the angiogenesis of endothelial cells (HUVECs) and this was inhibited by Curcumin-Veliparib combination. NECTIN-4 enhanced vascularization, induced vasodilation and triggered the angiogenic sprouting via endothelial tip cell filopodia. Data indicated that NECTIN-4 mediated angiogenesis is associated with PI3K-AKT-mediated nitric oxide (NO) formation. A noticeable increase in the NO enhanced epithelial NO level through HIF-1α mediated iNOS activation. We observed that increased NO enhanced the NECTIN-4 mediated eNOS expression and thereby elicited further angiogenesis. Curcumin antagonised the NECTIN-4-induced angiogenesis through inhibition of PI3K-AKT mediated eNOS pathway and Veliparib synergized the effect of Curcumin. Our observations indicate that NO is cardinal in inducing NECTIN-4 mediated angiogenesis in H357 cells. Thus, Curcumin-Veliparib combination suppresses angiogenesis through deregulation of the PI3K-AKT-eNOS pathway downstream to the NECTIN-4.     

Epigenetic silencing of genes enhanced by collective role of reactive oxygen species and MAPK signaling downstream ERK/Snail axis: Ectopic application of hydrogen peroxide repress CDH1 gene by enhanced DNA methyltransferase activity in human breast cancer

Loss of E-cadherin and epithelial to mesenchymal transition (EMT) are key steps in cancer progression. Reactive oxygen species (ROS) play significant roles in cellular physiology and homeostasis. Roles of E-cadherin (CDH1), EMT and ROS are intriguingly illustrated in many cancers without focusing their collective concert during cancer progression. We report that hydrogen peroxide (H₂O₂) treatment modulate CDH1 gene expression by epigenetic modification(s). Sublethal dosage of H₂O₂ treatment decrease E-cadherin, increase DNMT1, HDAC1, Snail, Slug and enrich H3K9me3 and H3K27me3 in the CDH1 promoter. The effect of H₂O₂ was attenuated by ROS scavengers; NAC, lupeol and beta-sitosterol. DNMT inhibitor, AZA prevented the H₂O₂ induced promoter-CpG-island methylation of CDH1. Treatment of cells with U0126 (inhibitor of ERK) reduced the expression of DNMT1, Snail and Slug, increased CDH1. This implicates that CDH1 is synergistically repressed by histone methylation, DNA methylation and histone deacetylation mediated chromatin remodelling and activation of Snail and Slug through ERK pathway. Increased ROS leads to activation of epigenetic machineries and EMT activators Snail/Slug which in their course of action inactivates CDH1 gene and lack of E-cadherin protein promotes EMT in breast cancer cells. ROS and ERK signaling facilitate epigenetic silencing and support the fact that subtle increase of ROS above basal level act as key cell signaling molecules. Free radical scavengers, lupeol and beta-sitosterol may be tested for therapeutic intervention of breast cancer. This work broadens the amplitude of epigenome and open avenues for investigations on conjoint effects of canonical and intrinsic metabolite signaling and epigenetic modulations in cancer.     

Comparison of Selected Protein Levels in Tumour and Surgical Margin in a Group of Patients with Oral Cavity Cancer

Oral cavity cancer belongs to head-and-neck squamous cell carcinoma group. The purpose of the study was to assess the levels of certain proteins in a tumour and surgical margin in a group of patients with oral cavity cancer. The levels of DAPK1, MGMT, CDH1, SFRP1, SFRP2, RORA, TIMP3, p16, APC and RASSF1 proteins were measured by ELISA in tissue homogenates. The protein levels of DAPK1, MGMT, CDH1, SFRP2 and RASSF1 were significantly higher in tumour tissue than in the margin, contrary to TIMP3 which was lower in the tumour itself. DAPK1 level in the tumour was significantly higher in females than in males, the MGMT and p16 levels were lower in the tumours with lymph node metastasis (N1 + N2) than in N0 samples. The CDH1 expression was higher in a group with smoking habits, whereas TIMP3 was lower in this group. Changes in the levels of proteins in tumour and surgical margin may be either reflective of tumour occurrence and development, or they might be also responsible for the progress and reoccurrence of the disease. Levels of the studied proteins might be good prognostic factors; however, further studies are required.     

SMAD signaling and redox imbalance cooperate to induce prostate cancer cell dormancy

Metastasis involves the dissemination of single or small clumps of cancer cells through blood or lymphatic vessels and their extravasation into distant organs. Despite the strong regulation of metastases development by a cell dormancy phenomenon, the dormant state of cancer cells remains poorly characterized due to the difficulty of in vivo studies. We have recently shown in vitro that clonogenicity of prostate cancer cells is regulated by a dormancy phenomenon that is strongly induced when cells are cultured both at low cell density and in a slightly hypertonic medium. Here, we characterized by RT-qPCR a genetic expression signature of this dormant state which combines the presence of both stemness and differentiation markers. We showed that both TFGβ/BMP signaling and redox imbalance are required for the full induction of this dormancy signature and cell quiescence. Moreover, reconstruction experiments showed that TFGβ/BMP signaling and redox imbalance are sufficient to generate a pattern of genetic expression displaying all characteristic features of the dormancy signature. Finally, we observed that low cell density was sufficient to activate TGFβ/BMP signaling and to generate a slight redox imbalance thus priming cells for dormancy that can be attained with a co-stimulus like hypertonicity, most likely through an increased redox imbalance. The identification of a dual regulation of dormancy provides a framework for the interpretation of previous reports showing a restricted ability of BMP signaling to regulate cancer cell dormancy in vivo and draws attention on the role of oxidative stress in the metastatic process.     

Specific Pyruvate Kinase M2 Inhibitor,Compound 3K,Induces Autophagic Cell Death through Disruption of the Glycolysis Pathway in Ovarian Cancer Cells

Ovarian cancer is a common cause of death among gynecological cancers. Although ovarian cancer initially responds to chemotherapy, frequent recurrence in patients remains a therapeutic challenge. Pyruvate kinase M2 (PKM2) plays a pivotal role in regulating cancer cell survival. However, its therapeutic role remains unclear. Here, we investigated the anticancer effects of compound 3K, a specific PKM2 inhibitor, on the regulation of autophagic and apoptotic pathways in SK-OV-3 (PKM2-overexpressing human ovarian adenocarcinoma cell line). The anticancer effect of compound 3K was examined using MTT and colony formation assays in SK-OV-3 cells. PKM2 expression was positively correlated with the severity of the tumor, and expression of pro-apoptotic proteins increased in SK-OV-3 cells following compound 3K treatment. Compound 3K induced AMPK activation, which was accompanied by mTOR inhibition. Additionally, this compound inhibited glycolysis, resulting in reduced proliferation of SK-OV-3 cells. Compound 3K treatment suppressed tumor progression in an in vivo xenograft model. Our findings suggest that the inhibition of PKM2 by compound 3K affected the Warburg effect and induced autophagic cell death. Therefore, use of specific PKM2 inhibitors to block the glycolytic pathway and target cancer cell metabolism represents a promising therapeutic approach for treating PKM2-overexpressing ovarian cancer.     

GAS6 Receptor Status Is Associated with Dormancy and Bone Metastatic Tumor Formation

Disseminated tumor cells (DTCs) are believed to lie dormant in the marrow before they can be activated to form metastases. How DTCs become dormant in the marrow and how dormant DTCs escape dormancy remains unclear. Recent work has shown that prostate cancer (PCa) cell lines express the growth-arrest specific 6 (GAS6) receptors Axl, Tyro3, and Mer, and become growth arrested in response to GAS6. We therefore hypothesized that GAS6 signaling regulates the proliferative activity of DTCs in the marrow. To explore this possibility, in vivo studies were performed where it was observed that when Tyro3 expression levels exceed Axl expression, the PCa cells exhibit rapid growth. When when Axl levels predominate, PCa cells remain largely quiescent. These findings suggest that a balance between the expression of Axl and Tyro3 is associated with a molecular switch between a dormant and a proliferative phenotype in PCa metastases.     

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.     

Anti-angiogenic effects of trabectedin (Yondelis; ET-743) on human breast cancer cells

Trabectedin, a tetrahydroisoquinoline alkaloid derived from a Caribbean tunicate Ecteinascidia turbinata, has been shown to have antitumor effects. In this study, we assessed the possible anti-angiogenic effects of trabectedin on human umbilical vein endothelial cells (HUVECs) and breast cancer cell lines. An XTT cell viability assay was used to determine cytotoxicity. A scratch assay was used to detect the migration of cells after trabectedin treatment. Angiogenic cytokine profiles of breast cancer cell lines, before and after treatment with trabectedin, were investigated using an angiogenesis antibody array. Changes in mRNA expression levels of VEGF were evaluated using qRT-PCR. Trabectedin inhibited the viability of HUVECs and breast cancer cells in a concentration- and time-dependent manner. The migration of both HUVECs and breast cancer cells was suppressed by trabectedin treatment. Angiogenic cytokines which are known to regulate tumorigenicity and angiogenesis, such as GM-CSF, IGFBP-2, VEGF, and uPA, were inhibited, while several anti-angiogenic cytokines such as TIMP-1 and Serpin E1were induced in breast cancer cells. Furthermore, expression levels of VEGF mRNA were inhibited in all breast cancer cells tested. Although additional studies are needed to elucidate the molecular mechanisms underlying the anti-angiogenic activity of trabectedin, our results suggest that trabectedin may act as a potential anti-angiogenic agent in breast cancer cells.