UCN-01 enhances cytotoxicity of irinotecan in colorectal cancer stem-like cells by impairing DNA damage response

Colorectal cancer (CRC) is one of the most common and lethal cancers worldwide. Despite recent progress, the prognosis of advanced stage CRC remains poor, mainly because of cancer recurrence and metastasis. The high morbidity and mortality of CRC has been recently ascribed to a small population of tumor cells that hold the potential of tumor initiation, i.e. cancer stem cells (CSCs), which play a pivotal role in cancer recurrence and metastasis and are not eradicated by current therapy. We screened CRC-SCs in vitro with a library of protein kinase inhibitors and showed that CRC-SCs are resistant to specific inhibition of the major signaling pathways involved in cell survival and proliferation. Nonetheless, broad-spectrum inhibition by the staurosporin derivative UCN-01 blocks CRC-SC growth and potentiates the activity of irinotecan in vitro and in vivo CRC-SC-derived models. Reverse-Phase Protein Microarrays (RPPA) revealed that, albeit CRC-SCs display individual phospho-proteomic profiles, sensitivity of CRC-SCs to UCN-01 relies on the interference with the DNA damage response mediated by Chk1. Combination of LY2603618, a specific Chk1/2 inhibitor, with irinotecan resulted in a significant reduction of CRC-SC growth in vivo, confirming that irinotecan treatment coupled to inhibition of Chk1 represents a potentially effective therapeutic approach for CRC treatment

MALAT1-dependent hsa_circ_0076611 regulates translation rate in triple-negative breast cancer

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (\u3b3-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (\u3b3-H2AXhigh /pATMhigh ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the \u3b3-H2AXhigh /pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The \u3b3-H2AXhigh /pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significanc

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1-S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (\uce\ub3-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (\uce\ub3-H2AXhigh/pATMhigh) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47\ue2\u80\u933.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20\ue2\u80\u933.58). The relationship between the \uce\ub3-H2AXhigh/pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The \uce\ub3-H2AXhigh/pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance

Expression of the Hippo transducer TAZ in association with WNT pathway mutations impacts survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

Background: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Methods: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). Results: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZpos/WNTmut) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZpos/WNTmutsignature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZpos/WNTmutsignature negatively impacted overall survival. Conclusions: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes

Expression of the Hippo transducer TAZ in association with WNT pathway mutations impacts survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy

Background: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Methods: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). Results: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZ pos /WNT mut ) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZ pos /WNT mut signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZ pos /WNT mut signature negatively impacted overall survival. Conclusions: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes

Growth Inhibition by the Tumor Suppressor p33ING1 in Immortalized and Primary Cells: Involvement of Two Silencing Domains and Effect of Ras

ING1 was identified as an inhibitor of growth and has been described as a tumor suppressor. Furthermore, the expression of ING1 is induced in senescent cells and antisense ING1 extends the proliferative life span of primary human fibroblasts. Cooperation of p33ING1 with p53 has been suggested to be an important function of ING1 in cell cycle control. Intriguingly, it has been shown that p33ING1 is associated with histone acetylation as well as with histone deacetylation function. Here we show that p33ING1 is a potent transcriptional silencer in various cell types. However, the silencing function is independent of the presence of p53. By use of deletion mutants two potent autonomous and transferable silencing domains were identified, but no evidence of an activation domain was found. The amino (N)-terminal silencing domain is sensitive to the histone deacetylase inhibitor trichostatin A (TSA) whereas the carboxy-terminal silencing function is resistant to TSA, suggesting that p33ING1 confers gene silencing through both HDAC-dependent and -independent mechanisms. Interestingly, the presence of oncogenic Ras, which is able to induce premature senescence, increases the p33ING1-mediated silencing function. Moreover, ING1-mediated silencing was reduced by coexpressing dominant-negative Ras or by treatment with the mitogen-activated protein kinase inhibitor PD98059 but not by treatment with SB203580, an inhibitor of the p38 pathway. In addition, we show that both silencing domains of ING1 are involved in cell cycle control, as measured by inhibition of colony formation of immortalized cells and by thymidine incorporation of primary human diploid fibroblasts (HDF). Interestingly, p33ING1 expression induces features of cellular senescence in HDFs

ChIP-on-Chip Analysis of In Vivo Mutant p53 Binding To Selected Gene Promoters

Growing evidence shows that mutant p53 proteins, which are present in many human tumors, gain oncogenic activities that can actively contribute to tumorigenesis. Mutant p53 proteins have been extensively shown to affect the expression of several genes involved in various aspects of cancer biology. We show here the ChIP-on-chip analysis of mutant p53 binding to a set of 154 promoters, composed of both validated and putative mutant p53 target genes. By using the chromatin obtained from mutant p53R175H-immunoprecipitation in proliferating SKBr3 breast cancer cells, we found that mutant p53 binds to 40 of the 154 promoters analyzed. siRNA-mediated mutant p53 knock-down modulates the transcript abundance of some of these target genes. Two-thirds of the mutant p53-bound promoters were also engaged by either p300 or PCAF acetyl-transferases, strongly indicating the presence of transcriptionally active complexes. We also found that NF-kB binding sites are overrepresented among the mutant p53-bound promoters; a ChIP-on-chip analysis confirmed that NF-kB p65 binds to 27 of the mutant p53-bound promoters, indicating that mutant p53 could influence the transcriptional output of these NF-kB target genes

The lncRNAMALAT1-WTAP axis: a novel layer of EMT regulation in hypoxic triple-negative breast cancer

Abstract Early metastatic disease development is one characteristic that defines triple-negative breast cancer (TNBC) as the most aggressive breast cancer (BC) subtype. Numerous studies have identified long non-coding RNAs (lncRNA) as critical players in regulating tumor progression and metastasis formation. Here, we show that MALAT1, a long non-coding RNA known to promote various features of BC malignancy, such as migration and neo angiogenesis, regulates TNBC cell response to hypoxia. By profiling MALAT1-associated transcripts, we discovered that lncRNA MALAT1 interacts with the mRNA encoding WTAP protein, previously reported as a component of the N6-methyladenosine (m6A) modification writer complex. In hypoxic conditions, MALAT1 positively regulates WTAP protein expression, which influences the response to hypoxia by favoring the transcription of the master regulators HIF1α and HIF1β. Furthermore, WTAP stimulates BC cell migratory ability and the expression of N-Cadherin and Vimentin, hallmarks of epithelial-to-mesenchymal transition (EMT). In conclusion, this study highlights the functional axis comprising MALAT1 and WTAP as a novel prognostic marker of TNBC progression and as a potential target for the development of therapeutic approaches for TNBC treatment