Anna Di Benedetto

High-mobility group A1 inhibits p53 by cytoplasmic relocalization of its proapoptotic activator HIPK2

High-mobility group A1 (HMGA1) overexpression and gene rearrangement are frequent events in human cancer, but the molecular basis of HMGA1 oncogenic activity remains unclear. Here we describe a mechanism through which HMGA1 inhibits p53-mediated apoptosis by counteracting the p53 proapoptotic activator homeodomain-interacting protein kinase 2 (HIPK2). We found that HMGA1 overexpression promoted HIPK2 relocalization in the cytoplasm and inhibition of p53 apoptotic function, while HIPK2 overexpression reestablished HIPK2 nuclear localization and sensitivity to apoptosis. HIPK2 depletion by RNA interference suppressed the antiapoptotic effect of HMGA1, which indicates that HIPK2 is the target required for HMGA1 to repress the apoptotic activity of p53. Consistent with this process, a strong correlation among HMGA1 overexpression, HIPK2 cytoplasmic localization, and low spontaneous apoptosis index (comparable to that observed in mutant p53-carrying tumors) was observed in WT p53-expressing human breast carcinomas. Hence, cytoplasmic relocalization of HIPK2 induced by HMGA1 overexpression is a mechanism of inactivation of p53 apoptotic function that we believe to be novel.

A divergent role for estrogen receptor-beta in node-positive and node-negative breast cancer classified according to molecular subtypes: an observational prospective study

IntroductionEstrogen receptor-alpha (ER-α) and progesterone receptor (PgR) are consolidated predictors of response to hormonal therapy (HT). In contrast, little information regarding the role of estrogen receptor-beta (ER-β) in various breast cancer risk groups treated with different therapeutic regimens is available. In particular, there are no data concerning ER-β distribution within the novel molecular breast cancer subtypes luminal A (LA) and luminal B (LB), HER2 (HS), and triple-negative (TN).MethodsWe conducted an observational prospective study using immunohistochemistry to evaluate ER-β expression in 936 breast carcinomas. Associations with conventional biopathological factors and with molecular subtypes were analyzed by multiple correspondence analysis (MCA), while univariate and multivariate Cox regression analysis and classification and regression tree analysis were applied to determine the impact of ER-β on disease-free survival in the 728 patients with complete follow-up data.ResultsER-β evenly distributes (55.5%) across the four molecular breast cancer subtypes, confirming the lack of correlation between ER-β and classical prognosticators. However, the relationships among the biopathological factors, analyzed by MCA, showed that ER-β positivity is located in the quadrant containing more aggressive phenotypes such as HER2 and TN or ER-α/PgR/Bcl2- tumors. Kaplan-Meier curves and Cox regression analysis identified ER-β as a significant discriminating factor for disease-free survival both in the node-negative LA (P = 0.02) subgroup, where it is predictive of response to HT, and in the node-positive LB (P = 0.04) group, where, in association with PgR negativity, it conveys a higher risk of relapse.ConclusionOur data indicated that, in contrast to node-negative patients, in node-positive breast cancer patients, ER-β positivity appears to be a biomarker related to a more aggressive clinical course. In this context, further investigations are necessary to better assess the role of the different ER-β isophorms.

HER2 Protein and Gene Variation between Primary and Metastatic Breast Cancer: Significance and Impact on Patient Care

Purpose: To analyze HER2 status in primary breast cancer (PBC) compared with correspondent metachronous metastases and to investigate whether BC phenotype may be predictive of change in HER2 expression. Experimental Design: HER2 was investigated by immunohistochemistry, silver in situ hybridization (SISH), and FISH, in a series of 137 tumors, building up a tissue microarray to concurrently analyze each single PBC and metastatic (MBC) on the same slide. Results: HER2 status was discordant in 14 cases (10%): 12 negative in PBC and positive in metastases and two positive in PBC and negative in metastases (P = 0.04). These findings were confirmed by a PCR based test termed Multiplex Ligation-dependent Probe Amplification (MLPA). HER2 status changed in hormone receptor-positive BC more frequently than in negative ones (P = 0.002). In addition, we evaluated HER2 gene and chromosome 17 copy number by SISH in the 123 cases with unchanged HER2 status during progression. We found consistent HER2 gene copy number stability in the 100 nonamplified cases. Conversely, of the 23 amplified PBC, 13 (57%) demonstrated a significant increase in the HER2 gene and chromosome 17 copy number in their paired metastases (P = 0.01), as defined by SISH (k = 0.54, P < 0.0001) and MLPA. Patients who changed HER2 status from negative to positive, presented significant longer time to progression when treated with trastuzumab compared to those who were untreated (P = 0.04). Conclusions: When feasible, HER2 reassessment in metastatic lesions should be carefully taken into account, especially for metastases coming from primary hormone receptor-positive BC. Clin Cancer Res; 17(7); 2055–64. ©2011 AACR.

The Cytoskeleton Regulatory Protein hMena (ENAH) Is Overexpressed in Human Benign Breast Lesions with High Risk of Transformation and Human Epidermal Growth Factor Receptor-2–Positive/Hormonal Receptor–Negative Tumors

Purpose: hMena (ENAH), a cytoskeleton regulatory protein involved in the regulation of cell motility and adhesion, is overexpressed in breast cancer. The aim of this study was to define at what stage of breast carcinogenesis hMena is overexpressed and to correlate hMena overexpression with established prognostic factors in breast cancer, focusing on human epidermal growth factor receptor-2 (HER-2). Experimental Design: hMena expression was assessed immunohistochemically in a prospective cohort of cases (n = 360) encompassing a highly representative spectrum of benign breast diseases associated with different risk of transformation, in situ, invasive, and metastatic tumors. Correlations with conventional pathologic and prognostic variables, such as proliferation index, hormonal receptor status, and HER-2 overexpression, were also evaluated. In vitro experiments were done to study the effect of neuregulin-1 and Herceptin treatments on hMena expression. Results: hMena protein is undetectable in normal breast and is weakly expressed in a small percentage of low-risk benign diseases (9%), but displays a progressive and significant increase of positivity in benign lesions at higher risk of transformation (slightly increased risk 43%; moderate increased risk 67%), in in situ (72%), invasive (93%), and metastatic breast cancer (91%). A significant direct correlation with tumor size (P = 0.04), proliferation index (P < 0.0001), and HER-2 overexpression (P < 0.0001) and an inverse relationship with estrogen (P = 0.036) and progesterone receptors (P = 0.001) are found in invasive carcinomas. In vitro experiments show that neuregulin-1 up-regulates, whereas Herceptin down-regulates, hMena expression. Conclusions: Our data provide new insights into the relevance of actin-binding proteins in human breast carcinogenesis and indicate hMena overexpression as a surrogate indicator in breast disease management.

miR‐10b*, a master inhibitor of the cell cycle, is down‐regulated in human breast tumours

Deregulated proliferation is a hallmark of cancer cells. Here, we show that microRNA‐10b* is a master regulator of breast cancer cell proliferation and is downregulated in tumoural samples versus matched peritumoural counterparts. Two canonical CpG islands (5 kb) upstream from the precursor sequence are hypermethylated in the analysed breast cancer tissues. Ectopic delivery of synthetic microRNA‐10b* in breast cancer cell lines or into xenograft mouse breast tumours inhibits cell proliferation and impairs tumour growth in vivo, respectively. We identified and validated in vitro and in vivo three novel target mRNAs of miR‐10b* (BUB1, PLK1 and CCNA2), which play a remarkable role in cell cycle regulation and whose high expression in breast cancer patients is associated with reduced disease‐free survival, relapse‐free survival and metastasis‐free survival when compared to patients with low expression. This also suggests that restoration of microRNA‐10b* expression might have therapeutic promise.

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.

Down-regulated nucleoside diphosphate kinase nm23-H1 expression is unrelated to high-risk human papillomavirus but associated with progression of cervical intraepithelial neoplasia and unfavourable prognosis in cervical cancer

Objective: One of the factors leading to an invasive phenotype is the nm23 family of metastases-associated genes. Of the six known members, nm23-H1 is the most frequently studied potential anti-metastatic gene in cervical cancer. However, the possible molecular links to oncogenic human papillomavirus (HPV) are completely unexplored as yet. Materials and methods: As a part of the HPV-Pathogen Istituto Superiore di Sanità study, a series of 150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions were examined by immunohistochemical staining for nm23-H1, and tested for HPV by polymerase chain reaction (PCR) with three sets of primers (MY09/11, GP5+/GP6+ and short PCR fragment). Follow-up data were available on all patients with SCC, and 67 CIN lesions were monitored by serial PCR for clearance or persistence of HPV after cone treatment. Results: A linear decrease (p = 0.001) was observed in nm23-H1 expression, starting from CIN1 (85% with normal expression), with the most dramatic down regulation on transition from CIN2 (70% normal) to CIN3 (39%) and further to SCC (25%). Reduced expression was associated with CIN3 or cancer at an odds ratio 8.72 (95% confidence interval 4.13 to 18.41). Nm23-H1 was of no use as a marker of the high-risk human papillomavirus (HR-HPV) type, and it did not predict clearance or persistence of HR-HPV after treatment of CIN. Importantly, nm23-H1 expression was a significant prognostic factor in cervical cancer, reduced expression being associated with lower survival (p = 0.022) in univariate analysis. In the multivariate (Cox) regression model, however, only the International Federation of Gynecology and Obstetrics stage (p = 0.001) and age (p = 0.011) remained independent prognostic predictors. Conclusions: Down-regulated nm23-H1 expression is markedly associated with progression from CIN2 to CIN3, and predicts poor prognosis in cervical cancer. Nm23-H1 down regulation is probably orchestrated by mechanisms independent of HR-HPV oncoproteins and is possibly related to the emergence of a proteolytic phenotype.

ATM kinase sustains HER2 tumorigenicity in breast cancer

ATM kinase preserves genomic stability by acting as a tumour suppressor. However, its identification as a component of several signalling networks suggests a dualism for ATM in cancer. Here we report that ATM expression and activity promotes HER2-dependent tumorigenicity in vitro and in vivo. We reveal a correlation between ATM activation and the reduced time to recurrence in patients diagnosed with invasive HER2-positive breast cancer. Furthermore, we identify ATM as a novel modulator of HER2 protein stability that acts by promoting a complex of HER2 with the chaperone HSP90, therefore preventing HER2 ubiquitination and degradation. As a consequence, ATM sustains AKT activation downstream of HER2 and may modulate the response to therapeutic approaches, suggesting that the status of ATM activity may be informative for the treatment and prognosis of HER2-positive tumours. Our findings provide evidence for ATMs tumorigenic potential revising the canonical role of ATM as a pure tumour suppressor.

Epidermal growth factor receptor gene copy number may predict lapatinib sensitivity in HER2-positive metastatic breast cancer

Objective: Lapatinib, a dual HER2/EGFR tyrosine kinase inhibitor (TKI), associated to capecitabine represents the treatment of choice in HER2-positive metastatic breast cancer (BC) patients in progression after trastuzumab-based therapy. Though lapatinib-based therapy prolongs the time to progression, its efficacy is often limited by the development of drug resistance. It is aimed to evaluate novel biomarkers predictive of lapatinib response, we analyzed EGFR protein and gene status in a series of 50 metastatic HER2-positive BC patients. Methods: Lapatinib was given at 1250 mg/day continuously and capecitabine at 2000 mg/m2/day every 3 weeks. EGFR protein expression and gene copy number (GCN) were assessed by immunohistochemistry and FISH, respectively. Receiver operating curve (ROC) analysis identified the value of > 3.36 EGFR copies/nucleus as the cut-off point able to discriminate responders versus non-responders. Results: A statistical significant correlation between EGFR GCN value > 3.36 and response to lapatinib (p = 0.01) was found. Cox regression analysis further supported these findings evidencing that HER2 score 3+ and EGFR GCN increase are positive predictor factors of lapatinib response. Conclusions: Though further investigations are needed to confirm these findings, EGFR GCN could be a suitable screening to identify the subset of BC patients particularly responsive to the dual TKI lapatinib.

The Hippo transducers TAZ/YAP and their target CTGF in male breast cancer

Male breast cancer (MBC) is a rare disease and its biology is poorly understood. Deregulated Hippo pathway promotes oncogenic functions in female breast cancer. We herein investigated the expression of the Hippo transducers TAZ/YAP and their target CTGF in MBC. Tissue microarrays containing samples from 255 MBC patients were immunostained for TAZ, YAP and CTGF. One hundred and twenty-nine patients were considered eligible. The Pearsons Chi-squared test of independence was used to test the association between categorical variables. The correlation between TAZ, YAP and CTGF was assessed with the Pearsons correlation coefficient. The Kaplan-Meier method and the log-rank test were used for estimating and comparing survival curves. Cox proportional regression models were built to identify variables impacting overall survival. Statistical tests were two-sided. Tumors were considered to harbor active TAZ/YAP-driven gene transcription when they co-expressed TAZ, or YAP, and CTGF. Patients whose tumors had the TAZ/CTGF and YAP/CTGF phenotypes experienced shorter overall survival compared with their negative counterparts (log rank p = 0.036 for both). TAZ/CTGF and YAP/CTGF tumors were associated with decreased survival in patients with invasive ductal carcinomas, G3 tumors, hormone receptor-positive tumors, and tumors with elevated Ki-67. Multivariate analyses confirmed that the TAZ/CTGF and YAP/CTGF phenotypes are independent predictors of survival (HR 2.03, 95% CI: 1.06–3.90, p = 0.033; and HR 2.00, 95% CI: 1.04–3.84, p = 0.037 respectively). Comparable results were obtained when excluding uncommon histotypes (TAZ/CTGF: HR 2.34, 95% CI: 1.16–4.73, p = 0.018. YAP/CTGF: HR 2.36, 95% CI: 1.17–4.77, p = 0.017). Overall, the TAZ/YAP-driven oncogenic program may be active in MBC, conferring poorer survival.