Cancer stem cell (SC) chemoresistance may be responsible for the poor clinical outcome of non-small-cell lung cancer (NSCLC) patients. In order to identify the molecular events that contribute to NSCLC chemoresistance, we investigated the DNA damage response in SCs derived from NSCLC patients. We found that after exposure to chemotherapeutic drugs NSCLC-SCs undergo cell cycle arrest, thus allowing DNA damage repair and subsequent cell survival. Activation of the DNA damage checkpoint protein kinase (Chk) 1 was the earliest and most significant event detected in NSCLC-SCs treated with chemotherapy, independently of their p53 status. In contrast, a weak Chk1 activation was found in differentiated NSCLC cells, corresponding to an increased sensitivity to chemotherapeutic drugs as compared with their undifferentiated counterparts. The use of Chk1 inhibitors in combination with chemotherapy dramatically reduced NSCLC-SC survival in vitro by inducing premature cell cycle progression and mitotic catastrophe. Consistently, the co-administration of the Chk1 inhibitor AZD7762 and chemotherapy abrogated tumor growth in vivo, whereas chemotherapy alone was scarcely effective. Such increased efficacy in the combined use of Chk1 inhibitors and chemotherapy was associated with a significant reduction of NSCLC-SCs in mouse xenografts. Taken together, these observations support the clinical evaluation of Chk1 inhibitors in combination with chemotherapy for a more effective treatment of NSCLC.

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 MET 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 MET 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 MET and might thus provide an additional option to inhibit this oncogene in tumors displaying its deregulation. [Cancer Res 2008;68(24):10128–36]

Fas/Apo-1 molecule, also designated as CD95, is a member of the TNF receptor family. Fas cross-linking by its natural ligand or by agonistic mAbs results in rapid induction of apoptosis in susceptible cells. in addition to the Fas full-length mRNA, human activated PBMC and tumor cell lines express several mRNA Fas variants that derive from alternative splicing of the primary transcript. All five variants identified, two of which are newly described here, code for soluble proteins that, with the exception of FasTMDel, are truncated in the extracytoplasmic region and possess short C-terminal amino acid sequences corresponding to a different reading frame. We have identified Abs that recognize all splicing variants and established a sandwich ELISA by which the soluble Fas molecules could be detected in culture supernatants of transfected cell lines and in PBMC following T cell activation. Next, we have studied in detail the functional role of these variants by apoptosis inhibition studies. We found that all soluble proteins block the apoptosis induced by either an agonistic Ab or, more importantly, by the natural Fas ligand in Fas-positive sensitive cell lines. interestingly, this functional property can be assigned to the first 49 amino acids of the mature protein that is the only region shared by the five soluble Fas molecules.