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High Mobility Group A1 (HMGA1) is an architectural chromatin factor involved in the regulation of gene expression and a master regulator in Triple Negative Breast Cancer (TNBC). In TNBC, HMGA1 is overexpressed and coordinates a gene network that controls cellular processes involved in tumour development, progression, and metastasis formation. Here, we find that the expression of HMGA1 and of the microtubule-destabilizing protein stathmin correlates in breast cancer (BC) patients. We demonstrate that HMGA1 depletion leads to a downregulation of stathmin expression and activity on microtubules resulting in decreased TNBC cell motility. We show that this pathway is mediated by the cyclin-dependent kinase inhibitor p27kip1 (p27). Indeed, the silencing of HMGA1 expression in TNBC cells results both in an increased p27 protein stability and p27-stathmin binding. When the expression of both HMGA1 and p27 is silenced, we observe a significant rescue in cell motility. These data, obtained in cellular models, were validated in BC patients. In fact, we find that patients with high levels of both HMGA1 and stathmin and low levels of p27 have a statistically significant lower survival probability in terms of relapse-free survival (RFS) and distant metastasis-free survival (DMFS) with respect to the patient group with low HMGA1, low stathmin, and high p27 expression levels. Finally, we show in an in vivo xenograft model that depletion of HMGA1 chemo-sensitizes tumour cells to paclitaxel, a drug that is commonly used in TNBC treatments. This study unveils a new interaction among HMGA1, p27, and stathmin that is critical in BC cell migration. Moreover, our data suggest that taxol-based treatments may be more effective in reducing the tumour burden when tumour cells express low levels of HMGA1.

Radiotherapy (RT) plus the anti‐EGFR monoclonal antibody Cetuximab (CTX) is an effective combination therapy for a subset of head and neck squamous cell carcinoma (HNSCC) patients. However, predictive markers of efficacy are missing, resulting in many patients treated with disappointing results and unnecessary toxicities. Here, we report that activation of EGFR upregulates miR‐9 expression, which sustains the aggressiveness of HNSCC cells and protects from RT‐induced cell death. Mechanistically, by targeting KLF5, miR‐9 regulates the expression of the transcription factor Sp1 that, in turn, stimulates tumor growth and confers resistance to RT+CTX in vitro and in vivo. Intriguingly, high miR‐9 levels have no effect on the sensitivity of HNSCC cells to cisplatin. In primary HNSCC, miR‐9 expression correlated with Sp1 mRNA levels and high miR‐9 expression predicted poor prognosis in patients treated with RT+CTX. Overall, we have discovered a new signaling axis linking EGFR activation to Sp1 expression that dictates the response to combination treatments in HNSCC. We propose that miR‐9 may represent a valuable biomarker to select which HNSCC patients might benefit from RT+CTX therapy., The combination therapy of Radiotherapy + Cetuximab (RT + CTX) is currently used for the treatment of HNSCC. Its lower toxicity compared to chemotherapy makes it the primary choice for fragile patients. This study identifies miR‐9 as a biomarker of RT + CTX responsiveness and explains why miR‐9 may be especially relevant in TP53 mutated HNSCC.

Epithelial ovarian cancer (EOC) is an infrequent but highly lethal disease, almost invariably treated with platinum‐based therapies. Improving the response to platinum represents a great challenge, since it could significantly impact on patient survival. Here, we report that silencing or pharmacological inhibition of CDK6 increases EOC cell sensitivity to platinum. We observed that, upon platinum treatment, CDK6 phosphorylated and stabilized the transcription factor FOXO3, eventually inducing ATR transcription. Blockage of this pathway resulted in EOC cell death, due to altered DNA damage response accompanied by increased apoptosis. These observations were recapitulated in EOC cell lines in vitro , in xenografts in vivo , and in primary tumor cells derived from platinum‐treated patients. Consistently, high CDK6 and FOXO3 expression levels in primary EOC predict poor patient survival. Our data suggest that CDK6 represents an actionable target that can be exploited to improve platinum efficacy in EOC patients. As CDK4/6 inhibitors are successfully used in cancer patients, our findings can be immediately transferred to the clinic to improve the outcome of EOC patients.

Platinum-based chemotherapy is the therapy of choice for epithelial ovarian cancer (EOC). Acquired resistance to platinum (PT) is a frequent event that leads to disease progression and predicts poor prognosis. To understand possible mechanisms underlying acquired PT-resistance, we have recently generated and characterized three PT-resistant isogenic EOC cell lines. Here, we more deeply characterize several PT-resistant clones derived from MDAH-2774 cells. We show that, in these cells, the increased PT resistance was accompanied by the presence of a subpopulation of multinucleated giant cells. This phenotype was likely due to an altered progression through the M phase of the cell cycle and accompanied by the deregulated expression of genes involved in M phase progression known to be target of mutant TP53. Interestingly, we found that PT-resistant MDAH cells acquired in the TP53 gene a novel secondary mutation (i.e., S185G) that accompanied the R273H typical of MDAH cells. The double p53S185G/R273H mutant increases the resistance to PT in a TP53 null EOC cellular model. Overall, we show how the selective pressure of PT is able to induce additional mutation in an already mutant TP53 gene in EOC and how this event could contribute to the acquisition of novel cellular phenotypes.

In epithelial ovarian cancer (EOC), response to platinum (PT)-based chemotherapy dictates subsequent treatments and predicts patients’ prognosis. Alternative splicing is often deregulated in human cancers and can be altered by chemotherapy. Whether and how changes in alternative splicing regulation could impact on the response of EOC to PT-based chemotherapy is still not clarified. We identified the splicing factor proline and glutamine rich (SFPQ) as a critical mediator of response to PT in an unbiased functional genomic screening in EOC cells and, using a large cohort of primary and recurrent EOC samples, we observed that it is frequently overexpressed in recurrent PT-treated samples and that its overexpression correlates with PT resistance. At mechanistic level, we show that, under PT treatment, SFPQ, in complex with p54nrb, binds and regulates the activity of the splicing factor SRSF2. SFPQ/p54nrb complex decreases SRSF2 binding to caspase-9 RNA, favoring the expression of its alternative spliced antiapoptotic form. As a consequence, SFPQ/p54nrb protects cells from PT-induced death, eventually contributing to chemoresistance. Overall, our work unveils a previously unreported SFPQ/p54nrb/SRSF2 pathway that in EOC cells plays a central role in regulating alternative splicing and PT-induced apoptosis and that could result in the design of new possible ways of intervention to overcome PT resistance.

Snail is a target of USP1 that links platinum response to metastasis in ovarian cancer., Resistance to platinum-based chemotherapy is a common event in patients with cancer, generally associated with tumor dissemination and metastasis. Whether platinum treatment per se activates molecular pathways linked to tumor spreading is not known. Here, we report that the ubiquitin-specific protease 1 (USP1) mediates ovarian cancer cell resistance to platinum, by regulating the stability of Snail, which, in turn, promotes tumor dissemination. At the molecular level, we observed that upon platinum treatment, USP1 is phosphorylated by ATM and ATR and binds to Snail. Then, USP1 de-ubiquitinates and stabilizes Snail expression, conferring resistance to platinum, increased stem cell–like features, and metastatic ability. Consistently, knockout or pharmacological inhibition of USP1 increased platinum sensitivity and decreased metastatic dissemination in a Snail-dependent manner. Our findings identify Snail as a USP1 target and open the way to a novel strategy to overcome platinum resistance and more successfully treat patients with ovarian cancer.

Recurrent, chemotherapy-resistant epithelial ovarian cancer (EOC) accounts for the most lethality among gynecologic cancers. Unfortunately, during the past 30 years, patients with EOC have experienced little improvement in overall survival, and standard treatment has not advanced beyond PT-based combination chemotherapy. Recent literature suggests that molecular and phenotypic associations exist between chemoresistance and the establishment of an epithelial-to-mesenchymal transition (EMT)-like program, by regulating the cancer cell plasticity and the tumor spreading. How this phenotype is linked to an alteration of the DNA damage response (DDR) activated by chemotherapy is still unclear and the possible molecular mediators connecting EMT and DDR are still undisclosed. Using an unbiased screening with shRNAs targeting genes belonging to apoptosis and DDR pathways, we identified ubiquitin specific protease-1 (USP1) as a key mediator of PT sensitivity in EOC. USP1 is a de-ubiquitinase previously implicated in the control of both DDR and EMT via the de-ubiquitination of FANCD2-FANCI and ID proteins, respectively. Here we report that USP1 impairment, using both genetic and pharmacologic approaches, significantly reduced PT-IC50 of different EOC cells encompassing the three most common histotypes (i.e., serous COV-362 and OVCAR8, endometrioid MDAH-2774,and clear cell TOV-21G). Moreover, USP1 regulates expression of several differentiation status-associated genes and controls spheroids formation in both untreated and PT-treated cells, the evasion from 3D-matrix, and the invasion of the mesothelial cell monolayer. Mechanistically, we identify the EMT transcription factor SNAIL as a new USP1 substrate. We demonstrated that upon PT treatment, USP1 is phosphorylated by the ATM and ATR kinases and this modification favors the binding of USP1 to SNAIL. USP1, by directly de-ubiquitinating SNAIL, stabilizes its expression and confers higher cell plasticity and resistance to PT-induced cell death. Accordingly, CRISPR-mediated USP1 knockout (KO) or its pharmacologic inhibition strongly decreased the ability of EOC cells to metastasize within the abdominal cavity and increased in vivo PT-induced cell death. Reintroduction of USP1 wild-type protein in USP1 KO cells restored their ability to spread in the abdomen and to resist PT therapy. Conversely, a USP1 mutant not phosphorylable by ATM or ATR failed to properly grow in the abdomen and was highly sensitive to PT treatment. Finally, we demonstrated that the ability of USP1 to regulate SNAIL is associated with a transcriptional program able to predict worse prognosis of EOC patients. Overall our work unveils several new relevant insights in the regulation of PT resistance in EOC. First, we report for the first time that a DUB is directly involved in the stabilization of an EMT transcription factor (i.e., SNAIL). Then we demonstrated that USP1 for its ability to link at molecular level EMT and DDR represents a particularly powerful oncoprotein that affects drug response and metastatic dissemination. Finally, we provide evidence that genetic and/or pharmacologic inhibition of USP1 could represent a promising strategy that might positively impact the management of EOC patients. Citation Format: Maura Sonego, Ilenia Pellarin, Alice Costa, Gian Luca Rampioni Vinciguerra, Michela Coan, Sara D’Andrea, Yohann Coutè, Riccardo Spizzo, Andrea Vecchione, Barbara Belletti, Monica Schiappacassi, Gustavo Baldassarre. Stabilization of SNAIL by USP-1 mediates chemoresistance and cell plasticity in epithelial ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A68.

The HMGA1 architectural transcription factor is an oncogene overexpressed in the vast majority of human cancers. HMGA1 is a highly connected node in the nuclear molecular network and the key aspect of HMGA1 involvement in cancer development is that HMGA1 simultaneously confers cells multiple oncogenic hits, ranging from global chromatin structural and gene expression modifications up to the direct functional alterations of key cellular proteins. Interestingly, HMGA1 also modulates DNA damage repair pathways. In this work, we provide evidences linking HMGA1 with Non-Homologous End Joining DNA repair. We show that HMGA1 is in complex with and is a substrate for DNA-PK. HMGA1 enhances Ligase IV activity and it counteracts the repressive histone H1 activity towards DNA ends ligation. Moreover, breast cancer cells overexpressing HMGA1 show a faster recovery upon induction of DNA double-strand breaks, which is associated with a higher survival. These data suggest that resistance to DNA-damaging agents in cancer cells could be partially attributed to HMGA1 overexpression thus highlighting the relevance of considering HMGA1 expression levels in the selection of valuable and effective pharmacological regimens.