<div>Abstract<p>MicroRNAs (miRNA) are a recently identified class of noncoding, endogenous, small RNAs that regulate gene expression, mainly at the translational level. These molecules play critical roles in several biological processes, such as cell proliferation and differentiation, development, and aging. It is also known that miRNAs play a role in human cancers where they can act either as oncogenes, down-regulating tumor suppressor genes, or as onco-suppressors, targeting molecules critically involved in promotion of tumor growth. One of such molecules is the tyrosine kinase receptor for hepatocyte growth factor, encoded by the <i>MET</i> oncogene. The MET receptor promotes a complex biological program named “invasive growth” that results from stimulation of cell motility, invasion, and protection from apoptosis. This oncogene is deregulated in many human tumors, where its most frequent alteration is overexpression. In this work, we have identified three miRNAs (miR-34b, miR-34c, and miR-199a*) that negatively regulate <i>MET</i> expression. Inhibition of these endogenous miRNAs, by use of antagomiRs, resulted in increased expression of MET protein, whereas their exogenous expression in cancer cells blocked MET-induced signal transduction and the execution of the invasive growth program, both in cells expressing normal levels of MET and in cancer cells overexpressing a constitutively active MET. Moreover, we show that these same miRNAs play a role in regulating the MET-induced migratory ability of melanoma-derived primary cells. In conclusion, we have identified miRNAs that behave as oncosuppressors by negatively targeting <i>MET</i> and might thus provide an additional option to inhibit this oncogene in tumors displaying its deregulation. [Cancer Res 2008;68(24):10128–36]</p></div>
Abstract Background: Micro RNAs (miRNAs) are short, non-coding RNA molecules that act as negative regulators of gene-expression, mainly at the post-transcriptional level. Alterations in miRNA functions have been implicated in a variety of human diseases, including cancer We demonstrated that the ectopic expression of miR-100 in cancer stem cells (CSCs) derived from aggressive, basal-like BC (HRs and HER2 negative) caused loss of stemness and the acquisition of a hormone receptor positive and endocrine treatment sensitive phenotype (Petrelli et al, Oncotarget 6;2315-30, 2015). We therefore sought to study whether miR-100 is a determinant of the endocrine-responsive phenotype in HR-positive BC patients (pts). Methods: Women with newly diagnosed, estrogen-receptor and/or progesterone-receptor positive, HER2 negative BC were eligible for this study. Treatment consisting of tamoxifen for pre-menopausal and letrozole for post-menopausal pts was administered daily for 21 days (+/- 3 days) before breast surgery. MiR-100 levels in pre-treatment tumor biopsies, measured as fold-change with respect to a reference RNA and transformed to the natural logarithms to normalize the data, were correlated with proliferative response to endocrine therapy, as measured by Ki67 expression in the final surgical specimen. The primary end-point was a complete proliferative response (CPR), defined as a post-treatment Ki67≤1%. Additionally, we considered a “post-hoc” composite endpoint where response was defined as a post treatment Ki67 <10% together with a Ki67 reduction ≥80% compared to pre-treatment values. The target accrual is 88 patients (pts). Here we report the results of the first interim analysis focusing on post-menopausal pts receiving letrozole. Results: A total of 42 pts were evaluable for miR-100 levels and response to endocrine therapy. Median ER and PgR expression was 99% (58%-99%) and 96% (0-99%) respectively. Median pre-treatment Ki67 was 18% (5-76%). Thirty-one tumors were ductal carcinomas, 9 were lobular and 2 were “other” histotypes. The median (range) miR-100 values in pre-treatment specimens was 2.253 (0.460-3.750). After treatment, median Ki67 was 4% (1%-46%) and the median percentage variation with respect to baseline values was -74% (0% to -94%). A CPR was observed in 5/42 pts (12%, 95% C.I. 5%-25%). The median miR-100 levels in responders and non-responders were 3.058 and 2.198, respectively (p = 0.03). Logistic regression analysis showed that each unit increase in miR-100 was associated with a 7-fold increase in the likelihood of a CPR (OR 7.056, 95% C.I. 1.103-45.141, p = 0.04). Considering the composite end-point, 17/42 pts (40%, 95% C.I. 27%-56%) were considered responders. Median miR-100 levels in responders and non-responders were 2.427 and 1.956, respectively (p = 0.05). Conclusions: preliminary results of this prospective clinical trial suggest that miR-100 can be a modulator of the endocrine-responsive phenotype in post-m pts with HR-positive breast cancer. The study is completing its target accrual and an investigation of miR-100 targets is being conducted. GZ and AP contributed equally to this work. Supported by Associazione Italiana per la Ricerca Sul Cancro (Investigator Grant IG-2013 Ref. 14451). Citation Format: Zucchini G, Petrelli A, Kubatzki F, Cargnelutti M, Di Virgilio MR, Sarotto I, Martincich L, Ponzone R, Martinello R, Sapino A, Nuzzo A, Giordano S, Montemurro F. Clinical evaluation of miR-100 as a predictor of endocrine-responsiveness in hormone-receptor positive breast cancer [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-09-21.
Abstract Hepatocellular carcinoma (HCC) is a multistage process but the nature of the molecular changes associated to the different steps, particularly the very early steps, is largely unknown. Recently, we have shown that activation of the Keap-1-NRF2 pathway is a very early event in the Resistant-Hepatocyte (R-H) rat model of hepatocarcinogenesis, suggesting that dysregulation of this pathway may have a causal role in HCC development. The aim of the present study was to investigate by Sanger fluorescence-based sequence analysis whether and when NRF2 mutations could occur in this experimental model. The results showed that mutations of NRF2 are extremely frequent (17/19; 90%) in early preneoplastic lesions positive for the stem/progenitor cell marker KRT-19, which are considered to be the precursor cell population of HCC in this model. Notably, all the mutations were missense and involved the Keap1-NRF2 binding regions. Mutations of Keap-1 were more rare and were found only in preneoplastic lesions lacking NRF2 mutations (10%). KRT-19 positive HCCs arising 1 year later also showed a high percentage of NRF2 mutation (> 50%), suggesting that mutation of this gene provides a selective advantage towards progression to HCC. Finally, preliminary studies in a subset of KRT-19 positive human HCCs, which are known to exhibit the worse prognosis, showed the presence of NRF2 mutations in a very high percentage (55%) of tumors. On the opposite, no mutations were detected in KRT-19 negative human HCCs. Conclusion: Our results demonstrate that mutations of NRF2 are highly frequent in a subset of human HCCs (KRT-19+) characterized by poor prognosis. Moreover, the findings stemming from the multistage carcinogenic rat model demonstrate that NRF2 mutation is a very early event, suggesting that dysregulation of the NRF2-Keap1 pathway is likely essential for the clonal expansion of KRT-19+ preneoplastic hepatocytes to HCC. Citation Format: Patrizia Zavattari, Andrea Perra, Marta A. Kowalik, Maddalena M. Angioni, Silvia Menegon, Annalisa Petrelli, Luca Quagliata, Giovanna M. Ledda-Columbano, Luigi Terraciano, Silvia Giordano, Amedeo Columbano. NRF2 mutations are frequent in early rat preneoplastic hepatic lesions and in human hepatocellular carcinomas positive for the stem/progenitor cell marker KRT-19. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1399. doi:10.1158/1538-7445.AM2014-1399
Abstract HER2 targeting with trastuzumab has changed the prognosis of breast cancer patients carrying amplification and/or overexpression of this oncogene. Despite this progress, however, resistance to trastuzumab occurs in the vast majority of patients. Newer anti-HER2 therapies, like the dual tyrosine-kinase inhibitor (TKI) lapatinib, show antitumor activity in a limited proportion of patients, indicating that HER2 can be still exploited as a target after trastuzumab failure. However, due to the high proportion of patients that fail to respond to these alternative strategies, it is reasonable to assume that cells escaping HER2 targeting may rely on alternative pathways not involving HER2 to sustain their growth. Their knowledge is relevant for exploiting new therapies. To investigate this hypothesis we generated a human HER2 overexpressing breast cancer cell line resistant to trastuzumab and lapatinib (T100) and we have characterized it genetically and molecularly. In T100 cells, the previously proposed mechanisms of resistance to HER2 inhibitors did not explain cell escape. Notably, silencing HER2 by shRNA did not affect the growth of T100 cells, suggesting loss of reliance upon HER2. Among the alternative pathways that we explored, altered levels of the antiapoptoptic proteins Mcl-1 and Survivin were observed. This suggested the possibility of targeting resistant cells with the multitarget inhibitor sorafenib. Moreover, sorafenib, nearly inactive in parental cells, strongly inhibited the in vitro growth of T100 cells. In conclusion, we provide a new model where the activation of HER2 independent mechanisms sustains the growth of tumor cells, significantly increasing the sensitivity to sorafenib. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A82.
