RASSF1A is a potential tumor suppressor gene that undergoes epigenetic inactivation in lung and breast cancers through hypermethylation of its promoter region.

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for ALDH1A1, ALDH3A1, and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed that most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH þ lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of

The mechanics of the microenvironment continuously modulates cell functions like growth, survival, apoptosis, differentiation, and morphogenesis via cytoskeletal remodeling and actomyosin contractility 1 – 3 . Although all these processes consume energy 4 , 5 , it is unknown if and how cells adapt their metabolic activity to variable mechanical cues. Here, we report that transfer of human bronchial epithelial cells (HBECs) from stiff to soft substrates causes downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by stress fiber disassembly, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small cell lung cancer cells (NSCLCs), which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress fiber population that is insensitive to substrate stiffness. In sum, our data unveil a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to attune energy production in variable microenvironments, while the resistance of the cytoskeleton to respond to mechanical cues allows high glycolytic rates to persist in cancer cells despite constant alterations of the tumor tissue.

We used CDK4/hTERT-immortalized normal human bronchial epithelial cells (HBECs) from several individuals to study lung cancer pathogenesis by introducing combinations of common lung cancer oncogenic changes (p53, KRAS, MYC) and followed the stepwise transformation of HBECs to full malignancy. This model demonstrated that: 1) the combination of five genetic alterations (CDK4, hTERT, sh-p53, KRASV12, and c-MYC) is sufficient for full tumorigenic conversion of HBECs; 2) genetically-identical clones of transformed HBECs exhibit pronounced differences in tumor growth, histology, and differentiation; 3) HBECs from different individuals vary in their sensitivity to transformation by these oncogenic manipulations; 4) high levels of KRASV12 are required for full malignant transformation of HBECs, however prior loss of p53 function is required to prevent oncogene-induced senescence; 5) over-expression of c-MYC greatly enhances malignancy but only in the context of sh-p53+KRASV12; 6) growth of parental HBECs in serum-containing medium induces differentiation while growth of oncogenically manipulated HBECs in serum increases in vivo tumorigenicity, decreases tumor latency, produces more undifferentiated tumors, and induces epithelial-to-mesenchymal transition (EMT); 7) oncogenic transformation of HBECs leads to increased sensitivity to standard chemotherapy doublets; 8) an mRNA signature derived by comparing tumorigenic vs. non-tumorigenic clones was predictive of outcome in lung cancer patients. Collectively, our findings demonstrate this HBEC model system can be used to study the effect of oncogenic mutations, their expression levels, and serum-derived environmental effects in malignant transformation, while also providing clinically translatable applications such as development of prognostic signatures and drug response phenotypes.

The ongoing unprecedented severe acute respiratory syndrome caused by the SARS-CoV-2 outbreak worldwide has highlighted the need for understanding viral-host interactions involved in mechanisms of virulence. Here, we show that the virulence factor Nsp1 protein of SARS-CoV-2 interacts with the host messenger RNA (mRNA) export receptor heterodimer NXF1-NXT1, which is responsible for nuclear export of cellular mRNAs. Nsp1 prevents proper binding of NXF1 to mRNA export adaptors and NXF1 docking at the nuclear pore complex. As a result, a significant number of cellular mRNAs are retained in the nucleus during infection. Increased levels of NXF1 rescues the Nsp1-mediated mRNA export block and inhibits SARS-CoV-2 infection. Thus, antagonizing the Nsp1 inhibitory function on mRNA export may represent a strategy to restoring proper antiviral host gene expression in infected cells.

Semaphorins SEMA3B and its homologue SEMA3F are 3p21.3 candidate tumor suppressor genes (TSGs), the expression of which is frequently lost in lung cancers. To test the TSG candidacy of SEMA3B and SEMA3F, we transfected them into lung cancer NCI-H1299 cells, which do not express either gene. Colony formation of H1299 cells was reduced 90% after transfection with wild-type SEMA3B compared with the control vector. By contrast, only 30 -40% reduction in colony formation was seen after the transfection of SEMA3F or SEMA3B variants carrying lung cancerassociated single amino acid missense mutations. H1299 cells transfected with wild-type but not mutant SEMA3B underwent apoptosis. We found that lung cancers (n ‫؍‬ 34) always express the neuropilin-1 receptor for secreted semaphorins, whereas 82% expressed the neuropilin-2 receptor. Because SEMA3B and SEMA3F are secreted proteins, we tested conditioned medium from COS-7 cells transfected with SEMA3B and SEMA3F and found that medium from wild-type SEMA3B transfectants reduced the growth of several lung cancer lines 30 -90%, whereas SEMA3B mutants or SEMA3F had little effect in the same assay. Sequencing of sodium bisulfite-treated DNA showed dense methylation of CpG sites in the SEMA3B 5 region of lung cancers not expressing SEMA3B but no methylation in SEMA3B-expressing tumors. These results are consistent with SEMA3B functioning as a TSG, the expression of which is inactivated frequently in lung cancers by allele loss and promoter region methylation.semaphorin ͉ neuropilin ͉ methylation T he semaphorin family comprised of secreted and membraneassociated proteins contributes to axonal path-finding during neural development by repulsing axons, inhibiting growth cone extension, and causing collapse of growth cones (1-3). The SEMA3 family members encode secreted proteins that signal through binding to neuropilin receptors (NP) interacting with plexins (1-3). Several semaphorins are expressed in adult nonneuronal tissues, suggesting other functions. For example, SEMA3A inhibited the motility of aortic endothelial cells expressing NP1, disrupted the formation of lamellipodia, induced depolymerization of F-actin (4), and inhibited branching morphogenesis in the fetal mouse lung (5). However, the roles of SEMA3B and SEMA3F in nonneuronal cells and human cancer are unknown.The loss of heterozygosity of chromosome 3p sequences is a critical event in the pathogenesis of lung and other cancers and directed a tumor suppressor gene (TSG) search to this region. Multiple distinct 3p regions are involved in human lung cancer pathogenesis including one at 3p21.3 where we identified 19 candidate TSGs (6, 7). This defined 3p21.3 region undergoes allele loss in Ϸ80% of primary lung cancers and Ϸ40% of preneoplastic or normal epithelial samples of smoking-damaged lung, marking it as one of the first sites involved (6). Two of the 19 genes are semaphorin family members (SEMA3B and SEMA3F) lying Ϸ70 kb apart (7,8). In assessing the TSG candidacy of SEMA3B and SEMA3F, we found only a few mutat...

BackgroundPromoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.Methods and FindingsIn an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.ConclusionsBy using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.

The common participation of oncogenic KRAS proteins in many of the most lethal human cancers, together with the ease of detecting somatic KRAS mutant alleles in patient samples, has spurred persistent and intensive efforts to develop drugs that inhibit KRAS activity1. However, advances have been hindered by the pervasive inter- and intra-lineage diversity in the targetable mechanisms that underlie KRAS-driven cancers, limited pharmacological accessibility of many candidate synthetic-lethal interactions and the swift emergence of unanticipated resistance mechanisms to otherwise effective targeted therapies. Here we demonstrate the acute and specific cell-autonomous addiction of KRAS-mutant non-small-cell lung cancer cells to receptor-dependent nuclear export. A multi-genomic, data-driven approach, utilizing 106 human non-small-cell lung cancer cell lines, was used to interrogate 4,725 biological processes with 39,760 short interfering RNA pools for those selectively required for the survival of KRAS-mutant cells that harbour a broad spectrum of phenotypic variation. Nuclear transport machinery was the sole process-level discriminator of statistical significance. Chemical perturbation of the nuclear export receptor XPO1 (also known as CRM1), with a clinically available drug, revealed a robust synthetic-lethal interaction with native or engineered oncogenic KRAS both in vitro and in vivo. The primary mechanism underpinning XPO1 inhibitor sensitivity was intolerance to the accumulation of nuclear IκBα (also known as NFKBIA), with consequent inhibition of NFκB transcription factor activity. Intrinsic resistance associated with concurrent FSTL5 mutations was detected and determined to be a consequence of YAP1 activation via a previously unappreciated FSTL5–Hippo pathway regulatory axis. This occurs in approximately 17% of KRAS-mutant lung cancers, and can be overcome with the co-administration of a YAP1–TEAD inhibitor. These findings indicate that clinically available XPO1 inhibitors are a promising therapeutic strategy for a considerable cohort of patients with lung cancer when coupled to genomics-guided patient selection and observation.