TRF2 positively regulates SULF2 expression increasing VEGF-A release and activity in tumor microenvironment.

The telomeric protein TRF2 is overexpressed in several human malignancies and contributes to tumorigenesis even though the molecular mechanism is not completely understood. By using a high-throughput approach based on the multiplexed Luminex X-MAP technology, we demonstrated that TRF2 dramatically affects VEGF-A level in the secretome of cancer cells, promoting endothelial cell-differentiation and angiogenesis. The pro-angiogenic effect of TRF2 is independent from its role in telomere capping. Instead, TRF2 binding to a distal regulatory element promotes the expression of SULF2, an endoglucosamine-6-sulfatase that impairs the VEGF-A association to the plasma membrane by inducing post-synthetic modification of heparan sulfate proteoglycans (HSPGs). Finally, we addressed the clinical relevance of our findings showing that TRF2/SULF2 expression is a worse prognostic biomarker in colorectal cancer (CRC) patients.

PLC-gamma-1 phosphorylation status is prognostic of metastatic risk in patients with early-stage Luminal-A and -B breast cancer subtypes.

BACKGROUND: Phospholipase Cγ1 (PLCγ1) is highly expressed in human tumours. Our previous studies reported that both stable and inducible PLCγ1 down-regulation can inhibit formation of breast-cancer-derived experimental lung metastasis. Further, high expression of PLCγ1 and its constitutively activated forms (i.e., PLCγ1-pY1253, PLCγ1-pY783) is associated with worse clinical outcome in terms of incidence of distant metastases, but not of local relapse in T1-T2, N0 breast cancer patients. METHODS: In the present retrospective study, we analysed the prognostic role of PLCγ1 in early breast cancer patients stratified according to the St. Gallen criteria and to their menopausal status. PLCγ1-pY1253 and PLCγ1-pY783 protein expression levels were determined by immunohistochemistry on tissue microarrays, and were correlated with patients' clinical data, using univariate and multivariate statistical analyses. RESULTS: In our series, the prognostic value of PLCγ1 overexpression was restricted to Luminal type tumours. From multivariate analyses, pY1253-PLCγ1High was an independent prognostic factor only in postmenopausal patients with Luminal-B tumours (hazard ratio [HR], 2.4; 95% confidence interval [CI], 1.1-5.3; P = 0.034). Conversely, PLCγ1-pY783High was a remarkably strong risk factor (HR, 20.1; 95% CI, 2.2-178.4; P = 0.003) for pre/perimenopausal patients with Luminal-A tumours. CONCLUSIONS: PLCγ1 overexpression is a strong predictive surrogate marker of development of metastases in early Luminal-A and -B breast cancer patients, being able to discriminate patients with high and low risk of metastases. Therefore, targeting the PLCγ1 pathway can be considered of potential benefit for prevention of metastatic disease.

SIRT6 interacts with TRF2 and promotes its degradation in response to DNA damage.

Telomere repeat binding factor 2 (TRF2) has been increasingly recognized to be involved in telomere maintenance and DNA damage response. Here, we show that TRF2 directly binds SIRT6 in a DNA independent manner and that this interaction is increased upon replication stress. Knockdown of SIRT6 up-regulates TRF2 protein levels and counteracts its down-regulation during DNA damage response, leading to cell survival. Moreover, we report that SIRT6 deactetylates in vivo the TRFH domain of TRF2, which in turn, is ubiquitylated in vivo activating the ubiquitin-dependent proteolysis. Notably, overexpression of the TRF2cT mutant failed to be stabilized by SIRT6 depletion, demonstrating that the TRFH domain is required for its post-transcriptional modification. Finally, we report an inverse correlation between SIRT6 and TRF2 protein expression levels in a cohort of colon rectal cancer patients. Taken together our findings describe TRF2 as a novel SIRT6 substrate and demonstrate that acetylation of TRF2 plays a crucial role in the regulation of TRF2 protein stability, thus providing a new route for modulating its expression level during oncogenesis and damage response.

CHK1-targeted therapy to deplete DNA replication-stressed, p53-deficient, hyperdiploid colorectal cancer stem cells.

OBJECTIVE: Cancer stem cells (CSCs) are responsible for tumour formation and spreading, and their targeting is required for tumour eradication. There are limited therapeutic options for advanced colorectal cancer (CRC), particularly for tumours carrying RAS-activating mutations. The aim of this study was to identify novel CSC-targeting strategies. DESIGN: To discover potential therapeutics to be clinically investigated as single agent, we performed a screening with a panel of FDA-approved or investigational drugs on primary CRC cells enriched for CSCs (CRC-SCs) isolated from 27 patients. Candidate predictive biomarkers of efficacy were identified by integrating genomic, reverse-phase protein microarray (RPPA) and cytogenetic analyses, and validated by immunostainings. DNA replication stress (RS) was increased by employing DNA replication-perturbing or polyploidising agents. RESULTS: The drug-library screening led to the identification of LY2606368 as a potent anti-CSC agent acting in vitro and in vivo in tumour cells from a considerable number of patients (∼36%). By inhibiting checkpoint kinase (CHK)1, LY2606368 affected DNA replication in most CRC-SCs, including RAS-mutated ones, forcing them into premature, lethal mitoses. Parallel genomic, RPPA and cytogenetic analyses indicated that CRC-SCs sensitive to LY2606368 displayed signs of ongoing RS response, including the phosphorylation of RPA32 and ataxia telangiectasia mutated serine/threonine kinase (ATM). This was associated with mutation(s) in TP53 and hyperdiploidy, and made these CRC-SCs exquisitely dependent on CHK1 function. Accordingly, experimental increase of RS sensitised resistant CRC-SCs to LY2606368. CONCLUSIONS: LY2606368 selectively eliminates replication-stressed, p53-deficient and hyperdiploid CRC-SCs independently of RAS mutational status. These results provide a strong rationale for biomarker-driven clinical trials with LY2606368 in patients with CRC.

Expression of ID4 protein in breast cancer cells induces reprogramming of tumour-associated macrophages.

BACKGROUND: As crucial regulators of the immune response against pathogens, macrophages have been extensively shown also to be important players in several diseases, including cancer. Specifically, breast cancer macrophages tightly control the angiogenic switch and progression to malignancy. ID4, a member of the ID (inhibitors of differentiation) family of proteins, is associated with a stem-like phenotype and poor prognosis in basal-like breast cancer. Moreover, ID4 favours angiogenesis by enhancing the expression of pro-angiogenic cytokines interleukin-8, CXCL1 and vascular endothelial growth factor. In the present study, we investigated whether ID4 protein exerts its pro-angiogenic function while also modulating the activity of tumour-associated macrophages in breast cancer. METHODS: We performed IHC analysis of ID4 protein and macrophage marker CD68 in a triple-negative breast cancer series. Next, we used cell migration assays to evaluate the effect of ID4 expression modulation in breast cancer cells on the motility of co-cultured macrophages. The analysis of breast cancer gene expression data repositories allowed us to evaluate the ability of ID4 to predict survival in subsets of tumours showing high or low macrophage infiltration. By culturing macrophages in conditioned media obtained from breast cancer cells in which ID4 expression was modulated by overexpression or depletion, we identified changes in the expression of ID4-dependent angiogenesis-related transcripts and microRNAs (miRNAs, miRs) in macrophages by RT-qPCR. RESULTS: We determined that ID4 and macrophage marker CD68 protein expression were significantly associated in a series of triple-negative breast tumours. Interestingly, ID4 messenger RNA (mRNA) levels robustly predicted survival, specifically in the subset of tumours showing high macrophage infiltration. In vitro and in vivo migration assays demonstrated that expression of ID4 in breast cancer cells stimulates macrophage motility. At the molecular level, ID4 protein expression in breast cancer cells controls, through paracrine signalling, the activation of an angiogenic programme in macrophages. This programme includes both the increase of angiogenesis-related mRNAs and the decrease of members of the anti-angiogenic miR-15b/107 group. Intriguingly, these miRNAs control the expression of the cytokine granulin, whose enhanced expression in macrophages confers increased angiogenic potential. CONCLUSIONS: These results uncover a key role for ID4 in dictating the behaviour of tumour-associated macrophages in breast cancer.

Expression of the Hippo transducer TAZ in association with WNT pathway mutations impacts survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy.

BACKGROUND: An extensive crosstalk co-regulates the Hippo and Wnt pathway. Preclinical studies revealed that the Hippo transducers YAP/TAZ mediate a number of oncogenic functions in gastric cancer (GC). Moreover, comprehensive characterization of GC demonstrated that the Wnt pathway is targeted by oncogenic mutations. On this ground, we hypothesized that YAP/TAZ- and Wnt-related biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. METHODS: In the present study, we included 86 patients with advanced GC treated with first-line chemotherapy in prospective phase II trials or in routine clinical practice. Tissue samples were immunostained to evaluate the expression of YAP/TAZ. Mutational status of key Wnt pathway genes (CTNNB1, APC and FBXW7) was assessed by targeted DNA next-generation sequencing (NGS). Survival curves were estimated and compared by the Kaplan-Meier product-limit method and the log-rank test, respectively. Variables potentially affecting progression-free survival (PFS) were verified in univariate Cox proportional hazard models. The final multivariate Cox models were obtained with variables testing significant at the univariate analysis, and by adjusting for all plausible predictors of the outcome of interest (PFS). RESULTS: We observed a significant association between TAZ expression and Wnt mutations (Chi-squared p = 0.008). Combined TAZ expression and Wnt mutations (TAZpos/WNTmut) was more frequently observed in patients with the shortest progression-free survival (negative outliers) (Fisher p = 0.021). Uni-and multivariate Cox regression analyses revealed that patients whose tumors harbored the TAZpos/WNTmut signature had an increased risk of disease progression (univariate Cox: HR 2.27, 95% CI 1.27-4.05, p = 0.006; multivariate Cox: HR 2.73, 95% CI 1.41-5.29, p = 0.003). Finally, the TAZpos/WNTmut signature negatively impacted overall survival. CONCLUSIONS: Collectively, our findings indicate that the oncogenic YAP/TAZ-Wnt crosstalk may be active in GC, conferring chemoresistant traits that translate into adverse survival outcomes.

Functional and prognostic significance of the genomic amplification of frizzled 6 (FZD6) in breast cancer.

Frizzled receptors mediate Wnt ligand signalling, which is crucially involved in regulating tissue development and differentiation, and is often deregulated in cancer. In this study, we found that the gene encoding the Wnt receptor frizzled 6 (FZD6) is frequently amplified in breast cancer, with an increased incidence in the triple-negative breast cancer (TNBC) subtype. Ablation of FZD6 expression in mammary cancer cell lines: (1) inhibited motility and invasion; (2) induced a more symmetrical shape of organoid three-dimensional cultures; and (3) inhibited bone and liver metastasis in vivo. Mechanistically, FZD6 signalling is required for the assembly of the fibronectin matrix, interfering with the organization of the actin cytoskeleton. Ectopic delivery of fibronectin in FZD6-depleted, triple-negative MDA-MB-231 cells rearranged the actin cytoskeleton and restored epidermal growth factor-mediated invasion. In patients with localized, lymph node-negative (early) breast cancer, positivity of tumour cells for FZD6 protein identified patients with reduced distant relapse-free survival. Multivariate analysis indicated an independent prognostic significance of FZD6 expression in TNBC tumours, predicting distant, but not local, relapse. We conclude that the FZD6-fibronectin actin axis identified in our study could be exploited for drug development in highly metastatic forms of breast cancer, such as TNBC. © 2016 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Association between AXL, Hippo Transducers, and Survival Outcomes in Male Breast Cancer.

Male breast cancer (MBC) is an uncommon malignancy. We have previously reported that the expression of the Hippo transducers TAZ/YAP and their target CTGF was associated with inferior survival in MBC patients. Preclinical evidence demonstrated that Axl is a transcriptional target of TAZ/YAP. Thus, we herein assessed AXL expression to further investigate the significance of active TAZ/YAP-driven transcription in MBC. For this study, 255 MBC samples represented in tissue microarrays were screened for AXL expression, and 116 patients were included. The association between categorical variables was verified by the Pearson's Chi-squared test of independence (2-tailed) or the Fisher Exact test. The relationship between continuous variables was tested with the Pearson's correlation coefficient. The Kaplan-Meier method was used for estimating survival curves, which were compared by log-rank test. Factors potentially impacting 10-year and overall survival were verified in Cox proportional regression models. AXL was positively associated with the TAZ/CTGF and YAP/CTGF phenotypes (P = 0.001 and P = 0.002, respectively). Patients with TAZ/CTGF/AXL- or YAP/CTGF/AXL-expressing tumors had inferior survival compared with non-triple-positive patients (log rank P = 0.042 and P = 0.048, respectively). The variables TAZ/CTGF/AXL and YAP/CTGF/AXL were adverse factors for 10-year survival in the multivariate Cox models (HR 2.31, 95%CI:1.02-5.22, P = 0.045, and HR 2.27, 95%CI:1.00-5.13, P = 0.050). Nearly comparable results were obtained from multivariate analyses of overall survival. The expression pattern of AXL corroborates the idea of the detrimental role of TAZ/YAP activation in MBC. Overall, Hippo-linked biomarkers deserve increased attention in this rare disease. J. Cell. Physiol. 232: 2246-2252, 2017. © 2016 Wiley Periodicals, Inc.

DNA damage repair and survival outcomes in advanced gastric cancer patients treated with first-line chemotherapy.

The DNA damage response (DDR) network is exploited by cancer cells to withstand chemotherapy. Gastric cancer (GC) carries deregulation of the DDR and harbors genetic defects that fuel its activation. The ATM-Chk2 and ATR-Chk1-Wee1 axes are deputed to initiate DNA repair. Overactivation of these pathways in cancer cells may represent an adaptive response for compensating genetic defects deregulating G1 -S transition (e.g., TP53) and ATM/ATR-initiated DNA repair (e.g., ARID1A). We hypothesized that DDR-linked biomarkers may predict clinical outcomes in GC patients treated with chemotherapy. Immunohistochemical assessment of DDR kinases (pATM, pChk2, pChk1 and pWee1) and DNA damage markers (γ-H2AX and pRPA32) was performed in biological samples from 110 advanced GC patients treated with first-line chemotherapy, either in phase II trials or in routine clinical practice. In 90 patients, this characterization was integrated with targeted ultra-deep sequencing for evaluating the mutational status of TP53 and ARID1A. We recorded a positive association between the investigated biomarkers. The combination of two biomarkers (γ-H2AXhigh /pATMhigh ) was an adverse factor for both progression-free survival (multivariate Cox: HR 2.23, 95%CI: 1.47-3.40) and overall survival (multivariate Cox: HR: 2.07, 95%CI: 1.20-3.58). The relationship between the γ-H2AXhigh /pATMhigh model and progression-free survival was consistent across the different TP53 backgrounds and was maintained in the ARID1A wild-type setting. Conversely, this association was no longer observed in an ARID1A-mutated subgroup. The γ-H2AXhigh /pATMhigh model negatively impacted survival outcomes in GC patients treated with chemotherapy. The mutational status of ARID1A, but apparently not TP53 mutations, affects its predictive significance.

Body mass index modifies the relationship between γ-H2AX, a DNA damage biomarker, and pathological complete response in triple-negative breast cancer.

BACKGROUND: Body mass index (BMI) is largely investigated as a prognostic and predictive factor in triple-negative breast cancer (TNBC). Overweight and obesity are linked to a variety of pathways regulating tumor-promoting functions, including the DNA damage response (DDR). The DDR physiologically safeguards genome integrity but, in a neoplastic background, it is aberrantly engaged and protects cancer cells from chemotherapy. We herein verified the role of BMI on a previously assessed association between DDR biomarkers and pathological complete response (pCR) in TNBC patients treated with neoadjuvant chemotherapy (NACT). METHODS: In this retrospective analysis 54 TNBC patients treated with NACT were included. The relationship between DDR biomarkers, namely phosphorylated H2A Histone Family Member X (γ-H2AX) and phosphorylated checkpoint kinase 1 (pChk1), and pCR was reconsidered in light of BMI data. The Pearson's Chi-squared test of independence (2-tailed) and the Fisher Exact test were employed to assess the relationship between clinical-molecular variables and pCR. Uni- and multivariate logistic regression models were used to identify variables impacting pCR. Internal validation was carried out. RESULTS: We observed a significant association between elevated levels of the two DDR biomarkers and pCR in patients with BMI < 25 (p = 0.009 and p = 0.022 for γ-H2AX and pChk1, respectively), but not in their heavier counterpart. Results regarding γ-H2AX were confirmed in uni- and multivariate models and, again, for leaner patients only (γ-H2AXhigh vs γ-H2AXlow: OR 10.83, 95% CI: 1.79-65.55, p = 0.009). The consistency of this finding was confirmed upon internal validation. CONCLUSIONS: The predictive significance of γ-H2AX varies according to BMI status. Indeed, elevated levels of γ-H2AX seemed associated with lower pCR rate only in leaner patients, whereas differences in pCR rate according to γ-H2AX levels were not appreciable in heavier patients. Larger investigations are warranted concerning the potential role of BMI as effect modifier of the relationship between DDR-related biomarkers and clinical outcomes in TNBC.

Neoadjuvant Sequential Docetaxel Followed by High-Dose Epirubicin in Combination With Cyclophosphamide Administered Concurrently With Trastuzumab. The DECT Trial.

To report the results of the DECT trial, a phase II study of locally advanced or operable HER2-positive breast cancer (BC) treated with taxanes and concurrent anthracyclines and trastuzumab. Eligible patients (stage IIA-IIIB HER2-positive BC, 18-75 years, normal organ functions, ECOG ≤1, and left ventricular ejection fraction (LVEF) ≥55%) received four cycles of neoadjuvant docetaxel, 100 mg/m(2) intravenously, plus trastuzumab 6 mg/kg (loading dose 8 mg/kg) every 3 weeks, followed by four 3-weekly cycles of epirubicin 120 mg/m(2) and cyclophosphamide, 600 mg/m(2) , plus trastuzumab. Primary objective was pathologic complete response (pCR) rate, defined as ypT0/is ypN0 at definitive surgery. We enrolled 45 consecutive patients. All but six patients (13.3%) completed chemotherapy and all underwent surgery. pCR was observed in 28 patients (62.2%) overall and in 6 (66.7%) from the inflammatory subgroup. The classification and regression tree analysis showed a 100% pCR rate in patients with BMI ≥25 and with hormone negative disease. The median follow up was 46 months (8-78). Four-year recurrence-free survival was 74.7% (95%CI, 58.2-91.2). Seven patients (15.6%) recurred and one died. Treatment was well tolerated, with limiting toxicity being neutropenia. No clinical cardiotoxicity was observed. Six patients (13.4%) showed a transient LVEF decrease (<10%). In one patient we observed a ≥10% asymptomatic LVEF decrease persisting after surgery. Notwithstanding their limited applicability due to the current guidelines, our findings support the efficacy of the regimen of interest in the neoadjuvant setting along with a fairly acceptable toxicity profile, including cardiotoxicity. Results on BMI may invite further assessment in future studies. J. Cell. Physiol. 231: 2541-2547, 2016. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc.

Anthropometric, metabolic and molecular determinants of human epidermal growth factor receptor 2 expression in luminal B breast cancer.

Genomic and trascriptomic profiling has recently contributed details to the characterization of luminal B breast cancer. We explored the contribution of anthropometric, metabolic, and molecular determinants to the multifaceted heterogeneity of this breast cancer subtype, with a specific focus on the association between body mass index (BMI), pre-treatment fasting glucose, hormone receptors, and expression of human epidermal growth factor receptor 2 (HER2). Extensively annotated specimens were obtained from 154 women with luminal B breast cancer diagnosed at two Italian comprehensive cancer centres. Participants' characteristics were descriptively analyzed overall and by HER2 status (positive vs. negative). BMI (<25 vs ≥25), pre-treatment fasting glucose (

The Hippo transducers TAZ and YAP in breast cancer: oncogenic activities and clinical implications.

The Hippo signalling is emerging as a tumour suppressor pathway whose function is regulated by an intricate network of intracellular and extracellular cues. Defects in the signal cascade lead to the activation of the Hippo transducers TAZ and YAP. Compelling preclinical evidence showed that TAZ/YAP are often aberrantly engaged in breast cancer (BC), where their hyperactivation culminates into a variety of tumour-promoting functions such as epithelial-to-mesenchymal transition, cancer stem cell generation and therapeutic resistance. Having acquired a more thorough understanding in the biology of TAZ/YAP, and the molecular outputs they elicit, has prompted a first wave of exploratory, clinically-focused analyses aimed at providing initial hints on the prognostic/predictive significance of their expression. In this review, we discuss oncogenic activities linked with TAZ/YAP in BC, and we propose clinical strategies for investigating their role as biomarkers in the clinical setting. Finally, we address the therapeutic potential of TAZ/YAP targeting and the modalities that, in our opinion, should be pursued in order to further study the biological and clinical consequences of their inhibition.

Splicing program of human MENA produces a previously undescribed isoform associated with invasive, mesenchymal-like breast tumors.

Human mena (hMENA), a member of the actin cytoskeleton regulators Ena/VASP, is overexpressed in high-risk preneoplastic lesions and in primary breast tumors and has been identified as playing a role in invasiveness and poor prognosis in breast cancers that express HER2. Here we identify a unique isoform, hMENAΔv6, derived from the hMENA alternative splicing program. In an isogenic model of human breast cancer progression, we show that hMENA(11a) is expressed in premalignant cells, whereas hMENAΔv6 expression is restricted to invasive cancer cells. "Reversion" of the malignant phenotype leads to concurrent down-regulation of all hMENA isoforms. In breast cancer cell lines, isoform-specific hMENA overexpression or knockdown revealed that in the absence of hMENA(11a), overexpression of hMENAΔv6 increased cell invasion, whereas overexpression of hMENA(11a) reduced the migratory and invasive ability of these cells. hMENA(11a) splicing was shown to be dependent on the epithelial regulator of splicing 1 (ESRP1), and forced expression of ESRP1 in invasive mesenchymal breast cancer cells caused a phenotypic switch reminiscent of a mesenchymal-to-epithelial transition (MET) characterized by changes in the cytoskeletal architecture, reexpression of hMENA(11a), and a reduction in cell invasion. hMENA-positive primary breast tumors, which are hMENA(11a)-negative, are more frequently E-cadherin low in comparison with tumors expressing hMENA(11a). These data suggest that polarized and growth-arrested cellular architecture correlates with absence of alternative hMENA isoform expression, and that the hMENA splicing program is relevant to malignant progression in invasive disease.

Gene status in HER2 equivocal breast carcinomas: impact of distinct recommendations and contribution of a polymerase chain reaction-based method.

BACKGROUND: The primary objectives of this study on carcinomas with equivocal HER2 expression were to assess the impact of distinct recommendations with regard to identifying patients eligible for anti-HER2 agents by fluorescence in situ hybridization (FISH) and to elucidate whether multiplex ligation-dependent probe amplification (MLPA) may be of support in assessing HER2 gene status. METHODS: A cohort of 957 immunohistochemistry-evaluated HER2-equivocal cases was analyzed by dual-color FISH. The results were assessed according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and American Society of Clinical Oncology (ASCO) and College of American Pathologists (CAP) 2007 and 2013 guidelines for dual- and single-signal in situ hybridization (ISH) assays. A subgroup of 112 cases was subjected to MLPA. RESULTS: HER2 amplification varied from 15% (ASCO/CAP 2007 HER2/CEP17 ratio) to 29.5% (FDA/EMA HER2 copy number). According to the ASCO/CAP 2013 interpretation of the dual-signal HER2 assay, ISH-positive carcinomas accounted for 19.7%. In contrast with the ASCO/CAP 2007 ratio, this approach labeled as positive all 32 cases (3.34%) with a HER2/CEP17 ratio <2 and an average HER2 copy number ≥6.0 signals per cell. In contrast, only one case showing a HER2 copy number <4 but a ratio ≥2 was diagnosed as positive. MLPA data correlated poorly with FISH results because of the presence of heterogeneous HER2 amplification in 33.9% of all amplified carcinomas; however, MLPA ruled out HER2 amplification in 75% of ISH-evaluated HER2-equivocal carcinomas. CONCLUSION: The ASCO/CAP 2013 guidelines seem to improve the identification of HER2-positive carcinomas. Polymerase chain reaction-based methods such as MLPA can be of help, provided that heterogeneous amplification has been ruled out by ISH.

Antiandrogen therapy in metastatic male breast cancer: results from an updated analysis in an expanded case series.

Male breast cancer is a rare disease treated as hormone receptor-positive female breast cancer. The characterization of breast cancer at the molecular level has lately revealed gender-related differences. As the androgen receptor is emerging as a potential oncogenic driver in male breast cancer, we analyzed efficacy data from metastatic patients treated with antiandrogens. We evaluated the activity of cyproterone acetate, either as a monotherapy or combined with a GnRH analog, in 36 metastatic male breast cancer patients. Fourteen patients were treated with cyproterone acetate as monotherapy and 22 patients with complete androgen blockade. We recorded 4 complete responses and 15 partial responses, for an overall response rate of 52.8 % (95 % CI, 36.5-69.4). Stable disease was reported in 11 patients. Median PFS was 8.9 months (95 % CI, 6.1-11.7), and median OS was 24.3 months (95 % CI, 22.5-26.1). Data on androgen receptor expression were available for 7 patients. All the 4 patients with androgen receptor-expressing tumors had a clinical benefit, including a patient with an estrogen receptor-negative disease. Conversely, none of the 3 patients with androgen receptor-negative tumors had a tumor response. Antiandrogen-based therapy showed efficacy in metastatic male breast cancer patients. Our results encourage considering antiandrogens in the therapeutic continuum, especially if supported by androgen receptor expression.

Outcomes of HER2-positive early breast cancer patients in the pre-trastuzumab and trastuzumab eras: a real-world multicenter observational analysis. The RETROHER study.

Addition of trastuzumab to adjuvant chemotherapy has dramatically reduced the risk of recurrence and has become the standard of care for human epidermal growth factor receptor 2 (HER2)-positive early breast cancer patients. Since most data on trastuzumab benefits come from clinical trials, conducted in selected patient populations, we performed a retrospective analysis of HER2-positive early breast cancer patients treated in the "pre-trastuzumab" and "trastuzumab" eras, with the aim to determine patients' outcomes in real-world practice. 925 consecutive HER2-positive breast cancer patients treated with adjuvant chemotherapy in ten Italian oncologic centers were identified. Patients who had received adjuvant chemotherapy alone (cohort A, 352 patients), and patients who had received adjuvant chemotherapy followed or combined with trastuzumab (cohort B, 573 patients) were analyzed. Relapse rate at 3 years, relapse-free survival, and overall survival were significantly more unfavorable in the cohort A than in the cohort B (p < 0.0001). In multivariate analysis, factors related to relapse were younger age, advanced stage at diagnosis, absence of hormonal and of trastuzumab therapy. The benefit derived from the addition of trastuzumab was independent of nodal status and hormonal receptors expression. A subgroup analysis including 163 "triple positive" tumors with high levels of estrogen and progesterone receptor (TP50) suggested that addition of trastuzumab to adjuvant chemotherapy and hormonal therapy did not translate into better outcomes. In our analysis, trastuzumab benefit was confirmed in all but a small subset of TP50 tumors subgroups. In this subset further investigations are needed.

Gain of function of mutant p53: the mutant p53/NF-Y protein complex reveals an aberrant transcriptional mechanism of cell cycle regulation.

This article investigates the mechanistic aspects of mutant p53 "gain of function" in response to DNA damage. We show that mutant forms of p53 protein interact with NF-Y. The expression of cyclin A, cyclin B1, cdk1, and cdc25C, as well as the cdk1-associated kinase activities, is upregulated after DNA damage, provoking a mutant p53/NF-Y-dependent increase in DNA synthesis. Mutant p53 binds NF-Y target promoters and, upon DNA damage, recruits p300, leading to histone acetylation. The recruitment of mutant p53 to the CCAAT sites is severely impaired upon abrogation of NF-YA expression. Endogenous NF-Y, mutant p53, and p300 proteins form a triple complex upon DNA damage. We demonstrate that aberrant transcriptional regulation underlies the ability of mutant p53 proteins to act as oncogenic factors.

Cancer stem cells: are they responsible for treatment failure?

Overcoming resistance to standard anticancer treatments represents a significant challenge. The interest regarding cancer stem cells, a cellular population that has the ability to self-renew and to propagate the tumor, was prompted by experimental evidence delineating the molecular mechanisms that are selectively activated in this cellular subset in order to survive chemotherapy. This has also stimulated combination strategies aimed at rendering cancer stem cells vulnerable to anticancer agents. Moreover, cancer stem cells offer a unique opportunity for modeling human cancers in mice, thus emerging as a powerful tool for testing novel drugs and combinations in a simulation of human disease. These novel animal models may lay the foundation for a new generation of clinical trials aimed at anticipating the benefit to patients of anticancer therapies.

Overexpression of activated phospholipase Cγ1 is a risk factor for distant metastases in T1-T2, N0 breast cancer patients undergoing adjuvant chemotherapy.

Phospholipase Cγ1 (PLCγ1) is highly expressed in several tumors. We have previously reported that both stable and inducible PLCγ1 down-regulation resulted in an almost complete inhibition of breast cancer-derived experimental lung metastasis formation. The aim of our study is to evaluate the association between the expression of PLCγ1 and of PLCγ1 phosphorylated at Tyr1253 (PLCγ1-pY1253) and at Tyr783 (PLCγ1-pY783) with the clinical outcome of patients with node negative, T1/T2 breast cancers. The study groups consisted of 292 (training set) and 122 (validation set) patients presenting with primary unilateral breast carcinoma (T1-T2), with no evidence of nodal involvement and distant metastases. PLCγ1, PLCγ1-pY1253 and PLCγ1-pY783 protein expression were assessed by immunohistochemistry on tissue microarrays and the results correlated with the clinical data using Kaplan-Meier curves and multivariate Cox regression analysis. Tumor cells while expressing variable proportions of cytoplasmic PLCγ1, express PLCγ1-pY1253 and PLCγ1-pY783 predominantly in the nucleus. High expression of PLCγ1, and of its activated forms, is associated with a worse clinical outcome in terms of incidence of distant metastases, and not of local relapse in T1-T2, N0 breast cancer patients undergone adjuvant chemotherapy. PLCγ1 over-expression appears to be a reliable predictive surrogate marker of development of metastases. Thus, targeting PLCγ1 pathways might represent a potential therapeutic approach for the prevention of metastatic disease in breast cancer.