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Lung cancer is the leading cause of cancer-associated death, with a global 5-year survival rate20%. Early metastasis and recurrence remain major challenges for lung cancer treatment. The stemness property of cancer cells has been suggested to play a key role in cancer plasticity, metastasis and drug-resistance, and is a potential target for drug development. In this study, we found that in non-small cell lung cancer (NSCLC), BMI1 and MCL1 play crucial roles of cancer stemness including invasion, chemo-resistance and tumour initiation. JNK signalling serves as a link between oncogenic pathway or genotoxicity to cancer stemness. The activation of JNK, either by mutant EGFR or chemotherapy agent, stabilized BMI1 and MCL1 proteins through suppressing the expression of E3-ubiquitin ligase HUWE1. In lung cancer patient samples, high level of BMI1 is correlated with poor survival, and the expression of BMI1 is positively correlated with MCL1. A novel small-molecule, BI-44, was developed, which effectively suppressed BMI1/MCL1 expressions and inhibited tumour formation and progression in preclinical models. Targeting cancer stemness mediated by BMI1/MCL1 with BI-44 provides the basis for a new therapeutic approach in NSCLC treatment.

Supplementary Figure 2 from Cancer-Targeted BikDD Gene Therapy Elicits Protective Antitumor Immunity against Lung Cancer

Supplementary Figure 1 from Cancer-Targeted BikDD Gene Therapy Elicits Protective Antitumor Immunity against Lung Cancer

This file contains figure legends.

This file contains technical details of experimental methods used in this study: 1. Colony formation assay; 2. Matrigel invasion assay; 3. Live cell migration assay; 4. Flow cytometry analysis; 5. Immunostaining; 6. ChIP-qPCR; 7. Cell proliferation assay; 8. Animal experiment.

Supplementary Figures S1-S4 and Supplementary Tables S1-S2.

Purpose: SOX9 is an important transcription factor required for development and has been implicated in several types of cancer. However, SOX9 has never been linked to lung cancer to date. Here, we show that SOX9 expression is upregulated in lung adenocarcinoma and show how it is associated with cancer cell growth.Experimental Design: Data mining with five microarray data sets containing 490 clinical samples, quantitative reverse transcription-PCR validation assay in 57 independent samples, and immunohistochemistry assay with tissue microarrays containing 170 lung tissue cores were used to profile SOX9 mRNA and protein expression. Short interference RNA suppression of SOX9 in cell lines was used to scrutinize functional role(s) of SOX9 and associated molecular mechanisms.Results: SOX9 mRNA and protein were consistently overexpressed in the majority of lung adenocarcinoma. Knockdown of SOX9 in lung adenocarcinoma cell lines resulted in marked decrease of adhesive and anchorage-independent growth in concordance with the upregulation of p21 (CDKN1A) and downregulation of CDK4. In agreement with higher SOX9 expression level in lung adenocarcinoma, the p21 mRNA level was significantly lower in tumors than that in normal tissues, whereas the opposite was true for CDK4, supporting the notion that SOX9 negatively and positively regulated p21 and CDK4, respectively. Finally, whereas SOX9-knockdown cells showed significantly attenuated tumorigenicity in mice, SOX9 transfectants consistently showed markedly stronger tumorigenicity.Conclusions: Our data suggest that SOX9 is a new hallmark of lung adenocarcinoma, in which SOX9 might contribute to gain of tumor growth potential, possibly acting through affecting the expression of cell cycle regulators p21 and CDK4. Clin Cancer Res; 16(17); 4363–73. ©2010 AACR.

CBLC (CBL proto-oncogene c) belongs to the CBL protein family, which has E3 ubiquitin ligase activity toward activated receptor tyrosine kinases. CBLC is frequently upregulated in non–small cell lung cancer (NSCLC), yet very little is known about the functions of CBLC in tumorigenesis. Here we show that CBLC is an epigenetically demethylated target and its expression can be upregulated in NSCLC after treatment with the DNA methylation inhibitor 5′-azacytidine. Depletion of CBLC significantly inhibited cell viability and clonogenicity in vitro and reduced tumor growth in a xenograft model. CBLC silencing further sensitized EGFR-mutated NSCLC cells to treatment with tyrosine kinase inhibitors. Conversely, ectopic expression of CBLC enhanced the activation of EGFR and downstream ERK1/2 signaling after ligand stimulation by competing with CBL for EGFR binding. Analysis of ubiquitin linkages on activated EGFR (aEGFR) revealed that CBLC ubiquitinated and positively regulated aEGFR stability through the conjugation of polyubiquitin by K6 and K11 linkages. This CBLC-mediated polyubiquitination promoted either preferential recycling of aEGFR back to the plasma membrane or trafficking to the cell nucleus. IHC analyses revealed a positive correlation between phospho-EGFR and CBLC in lung adenocarcinoma. In summary, we demonstrate a novel mechanism by which aEGFR escapes lysosomal degradation in a CBLC/ubiquitin-dependent manner to sustain its activation. Our work identifies CBLC as a potential diagnostic biomarker and also points to its utilization as a novel therapeutic target for NSCLC therapy.Significance: This work demonstrates the role of CBLC expression as a diagnostic biomarker and potential therapeutic target in lung adenocarcinoma. Cancer Res; 78(17); 4984–96. ©2018 AACR.

Cell differentiation within stem cell lineages can check proliferative potential, but nodal pathways that can limit tumor growth are obscure. Here, we report that lung cancer cell populations generate phenotypic and oncogenic plasticity via a switch between differentiation programs controlled by SOX2 and SOX9, thus altering proliferative and invasive capabilities. In lung cancer cells, SOX2 bound the EPCAM promoter to induce EpCAM–p21Cip1–cyclin A2 signaling, encouraging cell proliferation as well as barrier properties. In contrast, SOX9 bound the SLUG promoter to induce SLUG-mediated cell invasion with a spindle-like phenotype. Pharmacologic inhibition of HDAC elevated a SOX9-positive cell population from SOX2-positive cells, whereas ectopic expression of SOX2 inhibited SOX9 with increased H3K9me2 levels on the SOX9 promoter. In clinical specimens, the expression of SOX2 and SOX9 correlated negatively and positively with lung tumor grade, respectively. Our findings identify SOX2 and SOX9 as nodal epigenetic regulators in determining cancer cell plasticity and metastatic progression. Cancer Res; 76(23); 7036–48. ©2016 AACR.

Figure S1- CBLC expression is upregulated in NSCLC cell lines and clinical patients. Figure S2- Negative correlation between CBLC promoter methylation and mRNA expression in TCGA LUAD and LUSC studies.

This file contains following figures and tables: Fig. S1. Differential effects of cisplatin on SOX2- and SOX9-positive lung cancer cells (related to Fig. 1); Fig. S2. STR profiling in the derived stable clones (related to Fig. 2); Fig. S3. Expression switching between SOX2 and SOX9 (related to Fig. 2 and 3); Fig. S4. Ectopic expression of SOX2 attenuates SOX9 expression (related to Fig. 3); Fig. S5. Differential effects of SOX2 and SOX9 on cell proliferation (related to Fig. 3 and 4); Fig. S6. CCNA2-silencing attenuates cell growth in CL1-0 cells (related to Fig. 3); Fig. S7. SOX9-silencing attenuates lung invasion (related to Fig. 5); Fig. S8. Switching enrichment of H3K9me2 between CL1-1 and CL1-2 cells (related to Fig. 6); Fig. S9. Correlation analysis of SOX2 with SOX9, CCNA2 and HDAC1 in primary lung adenocarcinoma (related to Fig. 7); Fig. S10. Model for cancer cell plasticity (related to Fig. 1 to 7); Table S1. shRNA clones used in this study; Table S2. Primary antibodies used in this study; Table S3. Primers and probes used in this study; Table S4. Gene expression profiling data from the public domain used in this study.

MicroRNAs (miRNA) mediate distinct gene regulatory pathways triggered by epidermal growth factor receptor (EGFR) activation, which occurs commonly in lung cancers with poor prognosis. In this study, we report the discovery and mechanistic characterization of the miRNA miR-7 as an oncogenic “oncomiR” and its role as a key mediator of EGFR signaling in lung cancer cells. EGFR activation or ectopic expression of Ras as well as c-Myc stimulated miR-7 expression in an extracellular signal-regulated kinase (ERK)–dependent manner, suggesting that EGFR induces miR-7 expression through a Ras/ERK/Myc pathway. In support of this likelihood, c-Myc bound to the miR-7 promoter and enhanced its activity. Ectopic miR-7 promoted cell growth and tumor formation in lung cancer cells, significantly increasing the mortality of nude mice hosts, which were orthotopically implanted with lung cancers. Quantitative proteomic analysis revealed that miR-7 decreased levels of the Ets2 transcriptional repression factor ERF, the coding sequence of which was found to contain a miR-7 complementary sequence. Indeed, ectopic miR-7 inhibited production of ERF messages with a wild-type but not a silently mutated coding sequence, and ectopic miR-7 rescued growth arrest produced by wild-type but not mutated ERF. Together, these results identified that ERF is a direct target of miR-7 in lung cancer. Our findings suggest that miR-7 may act as an important modulator of EGFR-mediated oncogenesis, with potential applications as a novel prognostic biomarker and therapeutic target in lung cancer. Cancer Res; 70(21); 8822–31. ©2010 AACR.