Stefania Berton

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.

The Tumor Suppressor Functions of p27kip1 Include Control of the Mesenchymal/Amoeboid Transition

ABSTRACT In many human cancers, p27 downregulation correlates with a worse prognosis, suggesting that p27 levels could represent an important determinant in cell transformation and cancer development. Using a mouse model system based on v-src-induced transformation, we show here that p27 absence is always linked to a more aggressive phenotype. When cultured in three-dimensional contexts, v-src-transformed p27-null fibroblasts undergo a morphological switch from an elongated to a rounded cell shape, accompanied by amoeboid-like morphology and motility. Importantly, the acquisition of the amoeboid motility is associated with a greater ability to move and colonize distant sites in vivo. The reintroduction of different p27 mutants in v-src-transformed p27-null cells demonstrates that the control of cell proliferation and motility represents two distinct functions of p27, both necessary for it to fully act as a tumor suppressor. Thus, we highlight here a new p27 function in driving cell plasticity that is associated with its C-terminal portion and does not depend on the control of cyclin-dependent kinase activity.

p27Kip1 expression inhibits glioblastoma growth, invasion, and tumor-induced neoangiogenesis

The tumor suppressor gene CDKN1B encodes for a 27-kDa cyclin-dependent kinase inhibitory protein, p27Kip1, which together with its well-established role in the inhibition of cell proliferation, displays additional activities in the control of gene transcription and cell motility. p27Kip1 thus represents a good candidate for a gene therapy approach, especially in those cancers refractory to the conventional therapies, like human glioblastoma. Here, we show that overexpression of p27Kip1 in glioblastoma cell lines induced cell cycle arrest and inhibition of cell motility through extracellular matrix substrates. The use of adenoviral vectors in the treatment of glioblastoma in vivo showed that p27Kip1 was able to block not only cancer cell growth but also local invasion and tumor-induced neoangiogenesis. The latter effect was due to the ability of p27 to impair both endothelial cell growth and motility, thus preventing proper vessel formation in the tumor. The block of neoangiogenesis depended on cytoplasmic p27Kip1 antimigratory activity and was linked to its ability to bind to and inhibit the microtubule-destabilizing protein stathmin. Our work provides the first evidence that a successful p27Kip1-based gene therapy is linked to tumor microenvironment modification, thus opening new perspectives to the use of gene therapy approaches for the treatment of refractory cancers. [Mol Cancer Ther 2008;7(5):1164–75]

p27kip1 controls H-Ras/MAPK activation and cell cycle entry via modulation of MT stability.

Significance Different functions have been ascribed to p27kip1, originally identified as a universal cyclin-dependent kinase (CDK) inhibitor, fundamental for the control of cell proliferation and tumor progression. Yet, not all p27 functions can be explained by its ability to bind and inhibit CDKs. Here, we demonstrate that p27kip1 controls cell cycle entry also through a CDK-independent function, by regulating microtubule stability. Following growth factor stimulation, p27kip1 prevents full activation of H-Ras, acting on its subcellular compartmentalization, eventually restraining the activation of the MAPK pathway. Our work provides additional understanding of the mechanisms regulating the cell cycle and anticipates potential implications in diseases characterized by deregulated proliferation, such as cancer. The cyclin-dependent kinase (CDK) inhibitor p27kip1 is a critical regulator of the G1/S-phase transition of the cell cycle and also regulates microtubule (MT) stability. This latter function is exerted by modulating the activity of stathmin, an MT-destabilizing protein, and by direct binding to MTs. We recently demonstrated that increased proliferation in p27kip1-null mice is reverted by concomitant deletion of stathmin in p27kip1/stathmin double-KO mice, suggesting that a CDK-independent function of p27kip1 contributes to the control of cell proliferation. Whether the regulation of MT stability by p27kip1 impinges on signaling pathway activation and contributes to the decision to enter the cell cycle is largely unknown. Here, we report that faster cell cycle entry of p27kip1-null cells was impaired by the concomitant deletion of stathmin. Using gene expression profiling coupled with bioinformatic analyses, we show that p27kip1 and stathmin conjunctly control activation of the MAPK pathway. From a molecular point of view, we observed that p27kip1, by controlling MT stability, impinges on H-Ras trafficking and ubiquitination levels, eventually restraining its full activation. Our study identifies a regulatory axis controlling the G1/S-phase transition, relying on the regulation of MT stability by p27kip1 and finely controlling the spatiotemporal activation of the Ras-MAPK signaling pathway.

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.

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

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.