Sara D'Andrea

Targeted intraoperative radiotherapy impairs the stimulation of breast cancer cell proliferation and invasion caused by surgical wounding

Purpose: After apparently successful excision of breast cancer, risk of local recurrence remains high mainly in the area surrounding the original tumor, indicating that wound healing processes may be implicated. The proportional reduction of this risk by radiotherapy does not depend on the extent of surgery, suggesting that radiotherapy, in addition to killing tumor cells, may influence the tumor microenvironment. Experimental Design: We studied how normal and mammary carcinoma cell growth and motility are affected by surgical wound fluids (WF), collected over 24 h following breast-conserving surgery in 45 patients, 20 of whom had received additional TARGeted Intraoperative radioTherapy (TARGIT), immediately after the surgical excision. The proteomic profile of the WF and their effects on the activation of intracellular signal transduction pathways of breast cancer cells were also analyzed. Results: WF stimulated proliferation, migration, and invasion of breast cancer cell lines. The stimulatory effect was almost completely abrogated when fluids from TARGIT-treated patients were used. These fluids displayed altered expression of several cytokines and failed to properly stimulate the activation of some intracellular signal transduction pathways, when compared with fluids harvested from untreated patients. Conclusions: Delivery of TARGIT to the tumor bed alters the molecular composition and biological activity of surgical WF. This novel antitumoral effect could, at least partially, explain the very low recurrence rates found in a large pilot study using TARGIT. It also opens a novel avenue for identifying new molecular targets and testing novel therapeutic agents.

Stathmin activity influences sarcoma cell shape, motility, and metastatic potential

The balanced activity of microtubule-stabilizing and -destabilizing proteins determines the extent of microtubule dynamics, which is implicated in many cellular processes, including adhesion, migration, and morphology. Among the destabilizing proteins, stathmin is overexpressed in different human malignancies and has been recently linked to the regulation of cell motility. The observation that stathmin was overexpressed in human recurrent and metastatic sarcomas prompted us to investigate stathmin contribution to tumor local invasiveness and distant dissemination. We found that stathmin stimulated cell motility in and through the extracellular matrix (ECM) in vitro and increased the metastatic potential of sarcoma cells in vivo. On contact with the ECM, stathmin was negatively regulated by phosphorylation. Accordingly, a less phosphorylable stathmin point mutant impaired ECM-induced microtubule stabilization and conferred a higher invasive potential, inducing a rounded cell shape coupled with amoeboid-like motility in three-dimensional matrices. Our results indicate that stathmin plays a significant role in tumor metastasis formation, a finding that could lead to exploitation of stathmin as a target of new antimetastatic drugs.

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.

Genetic characterization of p27(kip1) and stathmin in controlling cell proliferation in vivo.

The CDK inhibitor p27kip1 is a critical regulator of cell cycle progression, but the mechanisms by which p27kip1 controls cell proliferation in vivo are still not fully elucidated. We recently demonstrated that the microtubule destabilizing protein stathmin is a relevant p27kip1 binding partner. To get more insights into the in vivo significance of this interaction, we generated p27kip1 and stathmin double knock-out (DKO) mice. Interestingly, thorough characterization of DKO mice demonstrated that most of the phenotypes of p27kip1 null mice linked to the hyper-proliferative behavior, such as the increased body and organ weight, the outgrowth of the retina basal layer and the development of pituitary adenomas, were reverted by co-ablation of stathmin. In vivo analyses showed a reduced proliferation rate in DKO compared to p27kip1 null mice, linked, at molecular level, to decreased kinase activity of CDK4/6, rather than of CDK1 and CDK2. Gene expression profiling of mouse thymuses confirmed the phenotypes observed in vivo, showing that DKO clustered with WT more than with p27 knock-out tissue. Taken together, our results demonstrate that stathmin cooperates with p27kip1 to control the early phase of G1 to S phase transition and that this function may be of particular relevance in the context of tumor progression.

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

Radiotherapy-induced miR-223 prevents relapse of breast cancer by targeting the EGF pathway

In breast cancer (BC) patients, local recurrences often arise in proximity of the surgical scar, suggesting that response to surgery may have a causative role. Radiotherapy (RT) after lumpectomy significantly reduces the risk of recurrence. We investigated the direct effects of surgery and of RT delivered intraoperatively (IORT), by collecting irradiated and non-irradiated breast tissues from BC patients, after tumor removal. These breast tissue specimens have been profiled for their microRNA (miR) expression, in search of differentially expressed miR among patients treated or not with IORT. Our results demonstrate that IORT elicits effects that go beyond the direct killing of residual tumor cells. IORT altered the wound response, inducing the expression of miR-223 in the peri-tumoral breast tissue. miR-223 downregulated the local expression of epidermal growth factor (EGF), leading to decreased activation of EGF receptor (EGFR) on target cells and, eventually, dampening a positive EGF–EGFR autocrine/paracrine stimulation loop induced by the post-surgical wound-healing response. Accordingly, both RT-induced miR-223 and peri-operative inhibition of EGFR efficiently prevented BC cell growth and reduced recurrence formation in mouse models of BC. Our study uncovers unknown effects of RT delivered on a wounded tissue and prompts to the use of anti-EGFR treatments, in a peri-operative treatment schedule, aimed to timely treat BC patients and restrain recurrence formation.

Stathmin Is Dispensable for Tumor Onset in Mice

The microtubule-destabilizing protein stathmin is highly expressed in several types of tumor, thus deserving the name of oncoprotein 18. High levels of stathmin expression and/or activity favor the metastatic spreading and mark the most aggressive tumors, thus representing a realistic marker of poor prognosis. Stathmin is a downstream target of many signaling pathways, including Ras-MAPK, PI3K and p53, involved in both tumor onset and progression. We thus hypothesized that stathmin could also play a role during the early stages of tumorigenesis, an issue completely unexplored. In order to establish whether stathmin expression is necessary for tumor initiation, we challenged wild type (WT), stathmin heterozygous and stathmin knock-out (KO) mice with different carcinogens. Using well-defined mouse models of carcinogenesis of skin, bladder and muscle by the means of 7,12-dimethylbenz[α]antracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA), N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) and 3-methylcholanthrylene (3MC) treatments, respectively, we demonstrated that knock-out of stathmin has no impact on the onset of cancer in mice. No significant difference was noticed either when the Ras oncogene was mutated (skin carcinogenesis model) or when the p53 pathway was inactivated (bladder carcinomas and fibrosarcomas). Finally, we concomitantly impinged on p53 and Ras pathways, by generating WT and stathmin KO mouse embryo fibroblasts transformed with papilloma virus large T antigen (LgTAg) plus the K-RasG12V oncogene. In vivo growth of xenografts from these transformed fibroblasts did not highlight any significant difference depending on the presence or absence of stathmin. Overall, our work demonstrates that stathmin expression is dispensable for tumor onset, at least in mice, thus making stathmin a virtually exclusive marker of aggressive disease and a promising therapeutic target for advanced cancers.

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.

p27kip1 expression limits H-Ras-driven transformation and tumorigenesis by both canonical and non-canonical mechanisms.

The tumor suppressor protein p27Kip1 plays a pivotal role in the control of cell growth and metastasis formation. Several studies pointed to different roles for p27Kip1 in the control of Ras induced transformation, although no explanation has been provided to elucidate these differences. We recently demonstrated that p27kip1 regulates H-Ras activity via its interaction with stathmin. Here, using in vitro and in vivo models, we show that p27kip1 is an important regulator of Ras induced transformation. In H-RasV12 transformed cells, p27kip1 suppressed cell proliferation and tumor growth via two distinct mechanisms: 1) inhibition of CDK activity and 2) impairment of MT-destabilizing activity of stathmin. Conversely, in K-Ras4BV12 transformed cells, p27kip1 acted mainly in a CDK-dependent but stathmin-independent manner. Using human cancer-derived cell lines and primary breast and sarcoma samples, we confirmed in human models what we observed in mice. Overall, we highlight a pathway, conserved from mouse to human, important in the regulation of H-Ras oncogenic activity that could have therapeutic and diagnostic implication in patients that may benefit from anti-H-Ras therapies.