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Numerous studies have implicated changes in the Y chromosome in male cancers, yet few have investigated the biological importance of Y chromosome noncoding RNA. Here we identify a group of Y chromosome-expressed long noncoding RNA (lncRNA) that are involved in male non-small cell lung cancer (NSCLC) radiation sensitivity. Radiosensitive male NSCLC cell lines demonstrated a dose-dependent induction of linc-SPRY3-2/3/4 following irradiation, which was not observed in radioresistant male NSCLC cell lines. Cytogenetics revealed the loss of chromosome Y (LOY) in the radioresistant male NSCLC cell lines. Gain- and loss-of-function experiments indicated that linc-SPRY3-2/3/4 transcripts affect cell viability and apoptosis. Computational prediction of RNA binding proteins (RBP) motifs and UV-cross-linking and immunoprecipitation (CLIP) assays identified IGF2BP3, an RBP involved in mRNA stability, as a binding partner for linc-SPRY3-2/3/4 RNA. The presence of linc-SPRY3-2/3/4 reduced the half-life of known IGF2BP3 binding mRNA, such as the antiapoptotic HMGA2 mRNA, as well as the oncogenic c-MYC mRNA. Assessment of Y chromosome in NSCLC tissue microarrays and expression of linc-SPRY3-2/3/4 in NSCLC RNA-seq and microarray data revealed a negative correlation between the loss of the Y chromosome or linc-SPRY3-2/3/4 and overall survival. Thus, linc-SPRY3-2/3/4 expression and LOY could represent an important marker of radiotherapy in NSCLC. SIGNIFICANCE: This study describes previously unknown Y chromosome-expressed lncRNA regulators of radiation response in male NSCLC and show a correlation between loss of chromosome Y and radioresistance. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/19/4046/F1.large.jpg., (©2020 American Association for Cancer Research.)

Targeted therapies for cancer are inherently limited by the inevitable recurrence of resistant disease after initial responses. To define early molecular changes within residual tumor cells that persist after treatment, we analyzed drug-sensitive lung adenocarcinoma cell lines exposed to reversible or irreversible epidermal growth factor receptor (EGFR) inhibitors, alone or in combination with MET-kinase inhibitors, to characterize the adaptive response that engenders drug resistance. Tumor cells displaying early resistance exhibited dependence on MET-independent activation of BCL-2/BCL-XL survival signaling. Further, such cells displayed a quiescence-like state associated with greatly retarded cell proliferation and cytoskeletal functions that were readily reversed after withdrawal of targeted inhibitors. Findings were validated in a xenograft model, showing BCL-2 induction and p-STAT3[Y705] activation within the residual tumor cells surviving the initial antitumor response to targeted therapies. Disrupting the mitochondrial BCL-2/BCL-XL antiapoptotic machinery in early survivor cells using BCL-2 Homology Domain 3 (BH3) mimetic agents such as ABT-737, or by dual RNAi-mediated knockdown of BCL-2/BCL-XL, was sufficient to eradicate the early-resistant lung-tumor-cells evading targeted inhibitors. Similarly, in a xenograft model the preemptive cotreatment of lung tumor cells with an EGFR inhibitor and a BH3 mimetic eradicated early TKI-resistant evaders and ultimately achieved a more durable response with prolonged remission. Our findings prompt prospective clinical investigations using BH3-mimetics combined with targeted receptor kinase inhibitors to optimize and improve clinical outcomes in lung-cancer treatment., (©2011 AACR.)

Patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer derive significant clinical benefit from treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Secondary EGFR mutations such as EGFR T790M commonly lead to resistance to these agents, limiting their long-term efficacy. Irreversible EGFR inhibitors such as CL-387,785 can overcome resistance and are in clinical development, yet acquired resistance against these agents is anticipated. We carried out a cell-based, in vitro random mutagenesis screen to identify EGFR mutations that confer resistance to CL-387,785 using T790M-mutant H1975 lung adenocarcinoma cells. Mutations at several residues occurred repeatedly leading to functional resistance to CL-387,785. These variants showed uninhibited cell growth, reduced apoptosis, and persistent EGFR activation in the presence of CL-387,785 as compared with parental H1975 cells, thus confirming their role in resistance. A screen of alternative agents showed that both an alternative EGFR inhibitor and a cyclin-dependent kinase 4 inhibitor led to significant inhibition of cell growth of the resistant mutants, suggestive of potential alternative treatment strategies. These results identify novel mutations mediating resistance to irreversible EGFR inhibitors and reveal alternative strategies to overcome or prevent the development of resistance in EGFR-mutant non-small cell lung cancers.

c-Met receptor tyrosine kinase (RTK) has not been extensively studied in malignant pleural mesothelioma (MPM). In this study, c-Met was overexpressed and activated in most of the mesothelioma cell lines tested. Expression in MPM tissues by immunohistochemistry was increased (82%) in MPM in general compared with normal. c-Met was internalized with its ligand hepatocyte growth factor (HGF) in H28 MPM cells, with robust expression of c-Met. Serum circulating HGF was twice as high in mesothelioma patients as in healthy controls. There was a differential growth response and activation of AKT and extracellular signal-regulated kinase 1/2 in response to HGF for the various cell lines. Dose-dependent inhibition (IC50 < 2.5 micromol/L) of cell growth in mesothelioma cell lines, but not in H2052, H2452, and nonmalignant MeT-5A (IC50 > 10 micromol/L), was observed with the small-molecule c-Met inhibitor SU11274. Furthermore, migration of H28 cells was blocked with both SU11274 and c-Met small interfering RNA. Abrogation of HGF-induced c-Met and downstream signaling was seen in mesothelioma cells. Of the 43 MPM tissues and 7 cell lines, we have identified mutations within the semaphorin domain (N375S, M431V, and N454I), the juxtamembrane domain (T1010I and G1085X), and an alternative spliced product with deletion of the exon 10 of c-Met in some of the samples. Interestingly, we observed that the cell lines H513 and H2596 harboring the T1010I mutation exhibited the most dramatic reduction of cell growth with SU11274 when compared with wild-type H28 and nonmalignant MeT-5A cells. Ultimately, c-Met would be an important target for therapy against MPM.

Non-small cell lung cancer (NSCLC) is a difficult disease to treat. The c-Met receptor is an attractive potential target for novel therapeutic inhibition in human cancers. We provide strong evidence that c-Met is overexpressed, activated, and sometimes mutated in NSCLC cell lines and tumor tissues. Expression of c-Met was found in all (100%) of the NSCLC tumor tissues examined (n = 23) and most (89%) of the cell lines (n = 9). Sixty-one percent of tumor tissues strongly expressed total c-Met, especially adenocarcinoma (67%). Specific expression of phospho-Met (p-Met) [Y1003] and [Y1230/1234/1235] was seen by immunohistochemistry. p-Met expression was preferentially observed at the NSCLC tumor invasive fronts. c-Met alterations were identified within the semaphorin domain (E168D, L299F, S323G, and N375S) and the juxtamembrane domain (R988C, R988C + T1010I, S1058P, and alternative splice product skipping entire juxtamembrane domain) of a NSCLC cell line and adenocarcinoma tissues. We validated c-Met as potential therapeutic target using small interfering RNA down-regulation of the receptor expression by 50% to 60% in NSCLC cells. This led to inhibition of p-Met and phospho-AKT and up to 57.1 +/- 7.2% cell viability inhibition at 72 hours. The selective small molecule inhibitor of c-Met SU11274 inhibited cell viability in c-Met-expressing NSCLC cells. SU11274 also abrogated hepatocyte growth factor-induced phosphorylation of c-Met and its downstream signaling. Here, we provide first direct evidence by small interfering RNA targeting and small molecule inhibitor that c-Met is important in NSCLC biology and biochemistry. These results indicate that c-Met inhibition will be an important therapeutic strategy against NSCLC to improve its clinical outcome.

Small cell lung cancer (SCLC) is an aggressive cancer, and most patients present with cancer already spread beyond the lung. The receptor tyrosine kinase (RTK) c-MET has been implicated in various solid tumors, including SCLC, and is involved in mediating tumorigenesis, cell motility, scattering, invasion and metastasis. Mutations of c-Met have been described in renal papillary carcinoma and gastrointestinal cancers including hepatocellular carcinoma. The sequence of c-MET was examined for possible mutations in the 10 SCLC cell lines and 32 paired-SCLC/normal tissues. Novel c-MET alterations were identified among 3 of 10 separate SCLC cell lines and in 4 of 32 SCLC tumor tissue samples. These include two different c-MET missense mutations in the juxtamembrane (JM) domain (R988C found in NCI-H69 and H249 cell lines; and T1010I in SCLC tumor sample T31). Also, there are one Sema domain missense mutation (E168D in SCLC tumor sample T5), two-base-pair insertional mutations (IVS13- (52-53)insCT in both SCLC tumor samples T26 and T27) within the pre-JM intron 13, as well as an alternative transcript involving exon 10 (H128 cell line). c-MET receptors are expressed at various levels among the 10 SCLC cell lines studied (high expression: H69, H345, H510, and H526; medium-expression: H128 and H146; and low/no-expression: H82, H209, H249, and H446). The level of c-MET expression does not have any apparent correlation with presence or absence of mutations of c-MET in the cell lines. We show that the two identified JM mutations (R988C and T1010I), when introduced into the interleukin-3 (IL-3)-dependent BaF3 cell line, regulated cell proliferation resulting in a small but significant growth factor independence. When introduced into a SCLC cell line (H446, with minimal endogenous wild-type c-MET expression), the JM mutations also regulated cell morphology and adhesion, as well as causing enhanced tumorigenicity by both increases in focus-formation and soft-agar colony-formation assays. Both of the JM mutations also increased cell motility and migration evident in wound healing assay and time-lapse video-microscopy speed analysis. The JM mutations also altered the c-MET RTK signaling, resulting in preferentially increased constitutive tyrosine phosphorylation of various cellular proteins, including the key focal adhesion protein paxillin on tyrosine residue Y31 (first CRKL-binding site), correlating with increased motility. These results suggest a novel and unique role of the JM domain in c-MET signaling in SCLC with significant implications in cytoskeletal functions and metastatic potential. The novel JM gain-of-function somatic mutations described are the first to be reported in SCLC, and may be associated with a more aggressive phenotype. It would now be useful to study the inhibition of c-MET as a therapeutic target against SCLC.

The regulation of biological functions including cell growth, viability, migration, and adhesion of small cell lung cancer (SCLC) cells depends largely on the autocrine or paracrine stimulation of growth factor receptors and chemokine receptors. Stem cell factor (SCF) and its receptor c-Kit have been identified as important regulators of SCLC viability and are coexpressed in approximately 40-70% of SCLC specimens. In vitro, the inhibition of c-Kit tyrosine kinase activity by the small molecule tyrosine kinase inhibitor STI571 (Gleevec) abrogates cell growth. We have investigated the role of c-Kit and chemokine receptors in the regulation of cell migration and adhesion of SCLC cells. CXCR4, the chemokine receptor for stromal cell-derived factor-1alpha (SDF-1alpha), was found to be the major chemokine receptor commonly expressed in all of the 10 SCLC cell lines tested. SCF and SDF-1alpha increased cellular proliferation over a course of 72 h in both the c-Kit- and the CXCR4-positive NCI-H69 SCLC cell line. Recently, SDF-1alpha and CXCR4 have been shown to be important regulators of migration and metastasis in breast and ovarian cancer. We found that SDF-1alpha dramatically increased cell motility and adhesion in CXCR4-expressing NCI-H446 SCLC cells. In addition, SDF-1alpha altered cell morphology with increased formation of filopodia and neurite-like projections. In NCI-H69 SCLC cells, SCF and SDF-1alpha cooperatively induced morphological changes and activated downstream signaling pathways. Treatment of NCI-H69 cells with STI571 specifically inhibited the c-Kit signaling events of Akt and p70 S6 kinase, whereas SDF-1alpha-mediated activation of Akt or p70 S6 kinase was normal. In contrast, the phosphatidylinositol 3-kinase inhibitor, LY294002, prevented these cells from adhering and completely blocked SCF- and/or SDF-1alpha-induced Akt or p70 S6 kinase phosphorylation. These results demonstrate that the CXCR4 receptor is functionally expressed in SCLC cells and may, therefore, be involved in the pathogenesis of SCLC in vivo. Inhibition of both the CXCR4 and the c-Kit downstream events could be a promising therapeutic approach in SCLC.