Showing papers by "Joshua Armenia published in 2013"

Inhibition of breast cancer local relapse by targeting p70S6 kinase activity

Abstract: 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