Maura Sonego

A microRNA signature defines chemoresistance in ovarian cancer through modulation of angiogenesis

Epithelial ovarian cancer is the most lethal gynecologic malignancy; it is highly aggressive and causes almost 125,000 deaths yearly. Despite advances in detection and cytotoxic therapies, a low percentage of patients with advanced stage disease survive 5 y after the initial diagnosis. The high mortality of this disease is mainly caused by resistance to the available therapies. Here, we profiled microRNA (miR) expression in serous epithelial ovarian carcinomas to assess the possibility of a miR signature associated with chemoresistance. We analyzed tumor samples from 198 patients (86 patients as a training set and 112 patients as a validation set) for human miRs. A signature of 23 miRs associated with chemoresistance was generated by array analysis in the training set. Quantitative RT-PCR in the validation set confirmed that three miRs (miR-484, -642, and -217) were able to predict chemoresistance of these tumors. Additional analysis of miR-484 revealed that the sensitive phenotype is caused by a modulation of tumor vasculature through the regulation of the VEGFB and VEGFR2 pathways. We present compelling evidence that three miRs can classify the response to chemotherapy of ovarian cancer patients in a large multicenter cohort and that one of these three miRs is involved in the control of tumor angiogenesis, indicating an option in the treatment of these patients. Our results suggest, in fact, that blockage of VEGF through the use of an anti-VEGFA antibody may not be sufficient to improve survival in ovarian cancer patients unless VEGFB signaling is also blocked.

p27kip1 controls cell morphology and motility by regulating microtubule-dependent lipid raft recycling

ABSTRACT p27kip1 (p27) is an inhibitor of cyclin/cyclin-dependent kinase complexes, whose nuclear loss indicates a poor prognosis in various solid tumors. When located in the cytoplasm, p27 binds Op18/stathmin (stathmin), a microtubule (MT)-destabilizing protein, and restrains its activity. This leads to MT stabilization, which negatively affects cell migration. Here, we demonstrate that this p27 function also influences morphology and motility of cells immersed in three-dimensional (3D)matrices. Cells lacking p27 display a decrease in MT stability, a rounded shape when immersed in 3D environments, and a mesenchymal-amoeboid conversion in their motility mode. Upon cell contact to extracellular matrix, the decreased MT stability observed in p27 null cells results in accelerated lipid raft trafficking and increased RhoA activity. Importantly, cell morphology, motility, MT network composition, and distribution of p27 null cells were rescued by the concomitant genetic ablation of Stathmin, implicating that the balanced expression of p27 and stathmin represents a crucial determinant for cytoskeletal organization and cellular behavior in 3D contexts.

Stathmin regulates mutant p53 stability and transcriptional activity in ovarian cancer

Stathmin is a p53‐target gene, frequently overexpressed in late stages of human cancer progression. Type II High Grade Epithelial Ovarian Carcinomas (HG‐EOC) represents the only clear exception to this observation. Here, we show that stathmin expression is necessary for the survival of HG‐EOC cells carrying a p53 mutant (p53MUT) gene. At molecular level, stathmin favours the binding and the phosphorylation of p53MUT by DNA‐PKCS, eventually modulating p53MUT stability and transcriptional activity. Inhibition of stathmin or DNA‐PKCS impaired p53MUT–dependent transcription of several M phase regulators, resulting in M phase failure and EOC cell death, both in vitro and in vivo. In primary human EOC a strong correlation exists between stathmin, DNA‐PKCS, p53MUT overexpression and its transcriptional targets, further strengthening the relevance of the new pathway here described. Overall our data support the hypothesis that the expression of stathmin and p53 could be useful for the identification of high risk patients that will benefit from a therapy specifically acting on mitotic cancer cells.

P70S6 kinase mediates breast cancer cell survival in response to surgical wound fluid stimulation

In early breast cancer, local relapses represent a determinant and not simply an indicator of risk for distant relapse and death. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Relevance of PI3K/mTOR/p70S6K signaling in breast tumorigenesis is very well documented. However, the pathway/s involved in the process of breast cancer local relapse are not well understood. The ribosomal protein p70S6K has been implicated in breast cancer cell response to post‐surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. Here, we show that p70S6K activity is required for the survival of breast cancer cells challenged in “hostile” microenvironments. We found that impairment of p70S6K activity in breast cancer cells strongly decreased their tumor take rate in nude mice. In line with this observation, if cells were challenged to grow in anchorage independence or in clonogenic assay, growth of colonies was strongly dependent on an intact p70S6K signaling. This in vitro finding was particularly evident when breast cancer cells were grown in the presence of wound fluids harvested following surgery from breast cancer patients, suggesting that the stimuli present in the post‐surgical setting at least partially relied on activity of p70S6K to stimulate breast cancer relapse. From a mechanistic point of view, our results indicated that p70S6K signaling was able to activate Gli1 and up‐regulate the anti‐apoptotic protein Bcl2, thereby activating a survival response in breast cancer cells challenged in hostile settings. Our work highlights a previously poorly recognized function of p70S6K in preserving breast cancer cell survival, which could eventually be responsible for local relapse and opens the way to the design of new and more specific therapies aiming to restrain the deleterious effects of wound response.

Inhibition of breast cancer local relapse by targeting p70S6 kinase activity

Dear Editor, Breast cancer (BC) is the most common cancer among women worldwide. The high percentage of early breast cancer (EBC) at diagnosis has raised the necessity of acquiring a better control of local relapses (Demicheli et al., 2008; Benson et al., 2009). Surgery itself and the subsequent wound healing process may represent perturbing factors for local recurrence and metastasis development (Demicheli et al., 2008; Troester et al., 2009). Both clinical and experimental evidences support this hypothesis. Multicentricity is a hallmark for many BC, yet 90% of local recurrences occur at the same quadrant of the primary cancer (Benson et al., 2009). Accordingly, wound fluids (WF) drained from BC patients after surgery stimulate proliferation and invasion of BC cells in vitro (Tagliabue et al., 2003; Belletti et al., 2008). Our previous studies implicated the 70-kDa ribosomal protein S6 kinase (hereafter p70S6K) in the response of BC cells to surgery-derived stimuli (Belletti et al., 2008). p70S6K is a serine/threonine kinase and downstream target of mTOR (Fenton and Gout, 2011). Many data suggest that p70S6K is implicated in BC onset and/or progression. The chromosomal region 17q23 containing the p70S6K gene (RPS6KB1) is amplified in 10% of all primary BC, leading to p70S6K overexpression (Sinclair et al., 2003), correlating with poor prognosis (Maruani et al., 2012) and increased risk of locoregional recurrence (van der Hage et al., 2004). Despite these strong correlative observations, the role of p70S6K in the process of BC relapse has never been investigated, nor has p70S6K been exploited as a therapeutic target. To investigate whether p70S6K activation was functionally involved in the response of BC cells to post-surgical WF, we generated BC cell lines with impaired p70S6K activity, by overexpressing a kinase inactive mutant (p70KR) or silencing p70S6K expression (sh-p70) (Supplementary Figure S1A and B). As additional approaches, we used the specific p70S6K1 inhibitor PF-4708671 (hereafter PF) (Pearce et al., 2010) and the clinically approved mTOR inhibitor, the rapamycin analogue Temsirolimus (hereafter Tems) (Supplementary Figure S1C). Then,we designed an in vivo experimental model resembling the course of human BC (Figure 1A). MD-MB-231 BC cells were bilaterally injected in nude mice mammary fat pads (MFP). When primary tumors reached 150–200 mm3, masses were surgically removed under anesthesia. After recovering, mice were followed up to detect appearance of local relapse. Eight weeks after surgery, mice were sacrificed and mammary glands, recurrences (when present), and lymphnodes were collected (Figure 1A). Since impairment of p70S6K activity in BC cells gave rise to smaller tumors (Harrington et al., 2004; Supplementary Figure S2A and B), we injected 1 × 10 control (CTR) cells (left MFP) and 2 × 10 p70KR expressing cells (right MFP) in order to obtain, at surgery time, primary tumors of similar size (Figure 1B upper panels and Supplementary Figure S2C). p70S6K activity was efficiently downmodulated in primary tumors derived from p70KR MDA-MB-231 cells (Supplementary Figure S3). When control cells were injected, local relapse typically appeared at 4–8 weeks after surgery, with a recurrence rate of 64% (Figure 1B lower panels, C, and E; Supplementary Table S1). Strikingly, in mice injected with p70KR cells, the percentage of recurrence dramatically dropped to 18% (Figure 1B lower panels, C, and E; Supplementary Table S1). Tumor spreading to loco-regional lymphnodes was detected only ipsilaterally to MFPs injected with CTR cells (Figure 1C). Similar results were obtained using MDA-MB-231 cells stably silenced for p70S6K (Figure 1E; Supplementary Table S1). PCR analyses excluded the possibility that recurrences observed in CTR cells injected-MFPs were caused by p70KR-expressing cells attracted to the surgery site via circulation (Supplementary Figure S4A). Increase of S6 phosphorylation was consistently detected in all relapses with respect to paired primary tumors, further supporting the critical role played by p70S6K in local re-growth (Supplementary Figure S4B). To exploit the potential of therapeutically targeting p70S6K, we tested in vivo specific p70S6K1 inhibition using PF (Pearce et al., 2010) and inhibition of mTOR using Tems, in vivo. We bilaterally injected MDA-MB-231 cells and then designed a 3-day schedule of peri-operative treatment (Day 2 1, Day 0, and Day + 1 with respect to surgery, Figure 1A), in order to restrain p70S6K activity during the surgery-induced inflammatory response. While this schedule did not affect the size of primary tumors (Figure 1D), perioperative treatment with PF resulted in highly effective suppression of recurrences (64% in controls vs. 23% in PF 600 mg and 11% in PF 1200 mg). By contrast, Tems was partially effective when given at a lower dose and, surprisingly, ineffective or even harmful to the mouse when administered at a higher dose (64% in controls vs. 29% in Tems 300 mg and 67% of Tems 600 mg) (Figure 1E; Supplementary Table S1). Statistical analysis demonstrated that the treatment with higher dose of PF was significantly effective in protecting against local relapse (P1⁄4 0.01 in Logrank test; Hazard Ratio 7.5; 95% Confidence Interval 1.3– 11.4) and significantly more efficient than 428 | Journal of Molecular Cell Biology (2013), 5, 428–431 doi:10.1093/jmcb/mjt027 Published online July 29, 2013

SUMOylation regulates p27Kip1 stability and localization in response to TGFβ

Exposure of normal and tumor-derived cells to TGFβ results in different outcomes, depending on the regulation of key targets. The CDK inhibitor p27(Kip1) is one of these TGFβ targets and is essential for the TGFβ-induced cell cycle arrest. TGFβ treatment inhibits p27(Kip1) degradation and induces its nuclear translocation, through mechanisms that are still unknown. Recent evidences suggest that SUMOylation, a post-translational modification able to modulate the stability and subcellular localization of target proteins, critically modifies members of the TGFβ signaling pathway. Here, we demonstrate that p27(Kip1) is SUMOylated in response to TGFβ treatment. Using different p27(Kip1) point mutants, we identified lysine 134 (K134) as the residue modified by small ubiquitin-like modifier 1 (SUMO1) in response to TGFβ treatment. TGFβ-induced K134 SUMOylation increased protein stability and nuclear localization of both endogenous and exogenously expressed p27(Kip1). We observed that SUMOylation regulated p27(Kip1) binding to CDK2, thereby governing its nuclear proteasomal degradation through the phosphorylation of threonine 187. Importantly, p27(Kip1) SUMOylation was necessary for proper cell cycle exit following TGFβ treatment. These data indicate that SUMOylation is a novel regulatory mechanism that modulates p27(Kip1) function in response to TGFβ stimulation. Given the involvement of TGFβ signaling in cancer cell proliferation and invasion, our data may shed light on an important aspect of this pathway during tumor progression.

Common biological phenotypes characterize the acquisition of platinum-resistance in epithelial ovarian cancer cells

Standard of care for Epithelial Ovarian Cancer (EOC) patients relies on platinum-based therapy. However, acquired resistance to platinum occurs frequently and predicts poor prognosis. To understand the mechanisms underlying acquired platinum-resistance, we have generated and characterized three platinum-resistant isogenic EOC cell lines. Resistant cells showed 3-to 5- folds increase in platinum IC50. Cross-resistance to other chemotherapeutic agents commonly used in the treatment of EOC patients was variable and dependent on the cell line utilized. Gene expression profiling (GEP) of coding and non-coding RNAs failed to identify a common signature that could collectively explain the mechanism of resistance. However, we observed that all resistant cell lines displayed a decreased level of DNA platination and a faster repair of damaged DNA. Furthermore, all platinum resistant cell lines displayed a change in their morphology and a higher ability to grown on mesothelium. Overall, we have established and characterized three new models of platinum-resistant EOC cell lines that could be exploited to further dissect the molecular mechanisms underlying acquired resistance to platinum. Our work also suggests that GEP studies alone, at least when performed under basal culture condition, do not represent the optimal way to identify molecular alterations linked to DNA repair pathway defects.

CDK6 protects epithelial ovarian cancer from platinum‐induced death via FOXO3 regulation

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.

Biomarker analysis of the MITO2 phase III trial of first-line treatment in ovarian cancer: predictive value of DNA-PK and phosphorylated ACC

Background No biomarker is available to predict prognosis of patients with advanced ovarian cancer (AOC) and guide the choice of chemotherapy. We performed a prospective-retrospective biomarker study within the MITO2 trial on the treatment of AOC. Patients and methods: MITO2 is a randomised multicentre phase 3 trial conducted with 820 AOC patients assigned carboplatin/paclitaxel (carboplatin: AUC5, paclitaxel: 175 mg/m², every 3 weeks for 6 cycles) or carboplatin/PLD-pegylated liposomal doxorubicin (carboplatin: AUC5, PLD: 30 mg/m², every 3 weeks for 6 cycles) as first line treatment. Sixteen biomarkers (pathways of adhesion/invasion, apoptosis, transcription regulation, metabolism, and DNA repair) were studied in 229 patients, in a tissue microarray. Progression-free and overall survival were analysed with multivariable Cox model. Results After 72 months median follow-up, 594 progressions and 426 deaths were reported; there was no significant difference between the two arms in the whole trial. No biomarker had significant prognostic value. Statistically significant interactions with treatment were found for DNA-dependent protein kinase (DNA-PK) and phosphorylated acetyl-coenzymeA carboxylase (pACC), both predicting worse outcome for patients receiving carboplatin/paclitaxel. Conclusion These data show that in presence of DNA-PK or pACC overexpression, carboplatin/paclitaxel might be less effective than carboplatin/PLD as first line treatment of ovarian cancer patients. Further validation of these findings is warranted.

Abstract B056: p70S6K activity drives local relapse in breast cancer

For early breast cancer (EBC) patients local relapse represents what mostly influences disease outcome. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Surgery itself and the consequent process of wound healing have been proposed to stimulate local recurrences via pathway(s) still to be clarified. Our previous studies implicated p70S6K pathway in breast cancer cell response to post-surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. We designed an in vivo experimental model resembling the course of human BC, in which MDA-MB-231 breast cancer cells were bilaterally injected in nude mice mammary fat pads and, when primary tumors were grown, masses were surgically removed under anesthesia. After recovering, mice were followed up to detect appearance of local relapse. Using this model, we dissected the role of p70S6K during breast cancer growth and recurrence by modulating its activity using both genetic and pharmacologic approaches. Our results showed that p70S6K positively contributed to proliferation and survival programs in breast cancer cell lines. In vivo, the analysis of the impact of p70S6K on primary tumor growth highlighted that a robust p70S6K signaling was required for the initiation of the tumor masses. But, more importantly, interfering with p70S6K activity strongly impaired local relapse. A three-day schedule of peri-operative treatment using specific pharmacological inhibition of p70S6K1 was sufficient to reduce by 83% the rate of local recurrence. We showed that inhibition of p70S6K activity induced apoptosis in cells challenged to survive in a hostile environment, supporting the idea that p70S6K signaling is necessary for the survival of isolated cancer cells in breast microenvironment, following surgery. The significance of our results was confirmed in human EBC specimens, proving that p70S6K activity is robustly induced by surgery, also in human patients. Taken together, our results provide a biological rationale for peri-operative treatment targeting p70S6K pathway, directed to compensating the harmful consequences of surgery and to restraining local recurrence in early breast cancer patients. Citation Format: Ilenia Segatto, Stefania Berton, Maura Sonego, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. p70S6K activity drives local relapse in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B056.