Joshua Armenia

The long tail of oncogenic drivers in prostate cancer.

Comprehensive genomic characterization of prostate cancer has identified recurrent alterations in genes involved in androgen signaling, DNA repair, and PI3K signaling, among others. However, larger and uniform genomic analysis may identify additional recurrently mutated genes at lower frequencies. Here we aggregate and uniformly analyze exome sequencing data from 1,013 prostate cancers. We identify and validate a new class of E26 transformation-specific (ETS)-fusion-negative tumors defined by mutations in epigenetic regulators, as well as alterations in pathways not previously implicated in prostate cancer, such as the spliceosome pathway. We find that the incidence of significantly mutated genes (SMGs) follows a long-tail distribution, with many genes mutated in less than 3% of cases. We identify a total of 97 SMGs, including 70 not previously implicated in prostate cancer, such as the ubiquitin ligase CUL3 and the transcription factor SPEN. Finally, comparing primary and metastatic prostate cancer identifies a set of genomic markers that may inform risk stratification.Meta-analysis of exome sequencing data identifies new recurrently mutated driver genes for prostate cancer. Comparison of primary and metastatic tumors further identifies genomic markers for advanced prostate cancer that may inform risk stratification.

Comprehensive molecular characterization of salivary duct carcinoma reveals actionable targets and similarity to apocrine breast cancer

Purpose: Salivary duct carcinoma (SDC) is an aggressive salivary malignancy, which is resistant to chemotherapy and has high mortality rates. We investigated the molecular landscape of SDC, focusing on genetic alterations and gene expression profiles. Experimental Design: We performed whole-exome sequencing, RNA sequencing, and immunohistochemical analyses in 16 SDC tumors and examined selected alterations via targeted sequencing of 410 genes in a second cohort of 15 SDCs. Results: SDCs harbored a higher mutational burden than many other salivary carcinomas (1.7 mutations/Mb). The most frequent genetic alterations were mutations in TP53 (55%), HRAS (23%), PIK3CA (23%), and amplification of ERBB2 (35%). Most (74%) tumors had alterations in either MAPK (BRAF/HRAS/NF1) genes or ERBB2. Potentially targetable alterations based on supportive clinical evidence were present in 61% of tumors. Androgen receptor (AR) was overexpressed in 75%; several potential resistance mechanisms to androgen deprivation therapy (ADT) were identified, including the AR-V7 splice variant (present in 50%, often at low ratios compared with full-length AR) and FOXA1 mutations (10%). Consensus clustering and pathway analyses in transcriptome data revealed striking similarities between SDC and molecular apocrine breast cancer. Conclusions: This study illuminates the landscape of genetic alterations and gene expression programs in SDC, identifying numerous molecular targets and potential determinants of response to AR antagonism. This has relevance for emerging clinical studies of ADT and other targeted therapies in SDC. The similarities between SDC and apocrine breast cancer indicate that clinical data in breast cancer may generate useful hypotheses for SDC. Clin Cancer Res; 22(18); 4623–33. ©2016 AACR.

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.

Multiplexed immunofluorescence delineates proteomic cancer cell states associated with metabolism

The phenotypic diversity of cancer results from genetic and nongenetic factors. Most studies of cancer heterogeneity have focused on DNA alterations, as technologies for proteomic measurements in clinical specimen are currently less advanced. Here, we used a multiplexed immunofluorescence staining platform to measure the expression of 27 proteins at the single-cell level in formalin-fixed and paraffin-embedded samples from treatment-naive stage II/III human breast cancer. Unsupervised clustering of protein expression data from 638,577 tumor cells in 26 breast cancers identified 8 clusters of protein coexpression. In about one-third of breast cancers, over 95% of all neoplastic cells expressed a single protein coexpression cluster. The remaining tumors harbored tumor cells representing multiple protein coexpression clusters, either in a regional distribution or intermingled throughout the tumor. Tumor uptake of the radiotracer 18F-fluorodeoxyglucose was associated with protein expression clusters characterized by hormone receptor loss, PTEN alteration, and HER2 gene amplification. Our study demonstrates an approach to generate cellular heterogeneity metrics in routinely collected solid tumor specimens and integrate them with in vivo cancer phenotypes.

Prospective Genomic Profiling of Prostate Cancer Across Disease States Reveals Germline and Somatic Alterations That May Affect Clinical Decision Making

PURPOSE A long natural history and a predominant osseous pattern of metastatic spread are impediments to the adoption of precision medicine in patients with prostate cancer. To establish the feasibility of clinical genomic profiling in the disease, we performed targeted deep sequencing of tumor and normal DNA from patients with locoregional, metastatic non-castrate, and metastatic castration-resistant prostate cancer (CRPC). METHODS Patients consented to genomic analysis of their tumor and germline DNA. A hybridization capture-based clinical assay was employed to identify single nucleotide variations, small insertions and deletions, copy number alterations and structural rearrangements in over 300 cancer-related genes in tumors and matched normal blood. RESULTS We successfully sequenced 504 tumors from 451 patients with prostate cancer. Potentially actionable alterations were identified in DNA damage repair (DDR), PI3K, and MAP kinase pathways. 27% of patients harbored a germline or a somatic alteration in a DDR gene that may predict for response to PARP inhibition. Profiling of matched tumors from individual patients revealed that somatic TP53 and BRCA2 alterations arose early in tumors from patients who eventually developed metastatic disease. In contrast, comparative analysis across disease states revealed that APC alterations were enriched in metastatic tumors, while ATM alterations were specifically enriched in CRPC. CONCLUSION Through genomic profiling of prostate tumors representing the disease clinical spectrum, we identified a high frequency of potentially actionable alterations and possible drivers of disease initiation, metastasis and castration-resistance. Our findings support the routine use of tumor and germline DNA profiling for patients with advanced prostate cancer, for the purpose of guiding enrollment in targeted clinical trials and counseling families at increased risk of malignancy.

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.

ERF mutations reveal a balance of ETS factors controlling prostate oncogenesis

Half of all prostate cancers are caused by the TMPRSS2–ERG gene-fusion, which enables androgens to drive expression of the normally silent E26 transformation-specific (ETS) transcription factor ERG in prostate cells. Recent genomic landscape studies of such cancers have reported recurrent point mutations and focal deletions of another ETS member, the ETS2 repressor factor ERF. Here we show these ERF mutations cause decreased protein stability and mostly occur in tumours without ERG upregulation. ERF loss recapitulates the morphological and phenotypic features of ERG gain in normal mouse prostate cells, including expansion of the androgen receptor transcriptional repertoire, and ERF has tumour suppressor activity in the same genetic background of Pten loss that yields oncogenic activity by ERG. In the more common scenario of ERG upregulation, chromatin immunoprecipitation followed by sequencing indicates that ERG inhibits the ability of ERF to bind DNA at consensus ETS sites both in normal and in cancerous prostate cells. Consistent with a competition model, ERF overexpression blocks ERG-dependent tumour growth, and ERF loss rescues TMPRSS2–ERG-positive prostate cancer cells from ERG dependency. Collectively, these data provide evidence that the oncogenicity of ERG is mediated, in part, by competition with ERF and they raise the larger question of whether other gain-of-function oncogenic transcription factors might also inactivate endogenous tumour suppressors.

Regulation of the glucocorticoid receptor via a BET-dependent enhancer drives antiandrogen resistance in prostate cancer

In prostate cancer, resistance to the antiandrogen enzalutamide (Enz) can occur through bypass of androgen receptor (AR) blockade by the glucocorticoid receptor (GR). In contrast to fixed genomic alterations, here we show that GR-mediated antiandrogen resistance is adaptive and reversible due to regulation of GR expression by a tissue-specific enhancer. GR expression is silenced in prostate cancer by a combination of AR binding and EZH2-mediated repression at the GR locus, but is restored in advanced prostate cancers upon reversion of both repressive signals. Remarkably, BET bromodomain inhibition resensitizes drug-resistant tumors to Enz by selectively impairing the GR signaling axis via this enhancer. In addition to revealing an underlying molecular mechanism of GR-driven drug resistance, these data suggest that inhibitors of broadly active chromatin-readers could have utility in nuanced clinical contexts of acquired drug resistance with a more favorable therapeutic index.