Dasatinib induces DNA damage and activates DNA repair pathways leading to senescence in non-small cell lung cancer cell lines with kinase-inactivating BRAF mutations

Improved therapies are greatly needed for non-small cell lung cancer (NSCLC) that does not harbor targetable kinase mutations or translocations. We previously demonstrated that NSCLC cells that harbor kinase-inactivating BRAF mutations (^KIBRAF) undergo senescence when treated with the multitargeted kinase inhibitor dasatinib. Similarly, treatment with dasatinib resulted in a profound and durable response in a patient with ^KIBRAF NSCLC. However, no canonical pathways explain dasatinib-induced senescence in ^KIBRAF NSCLC. To investigate the underlying mechanism, we used 2 approaches: gene expression and reverse phase protein arrays. Both approaches showed that DNA repair pathways were differentially modulated between ^KIBRAF NSCLC cells and those with wild-type (WT) BRAF. Consistent with these findings, dasatinib induced DNA damage and activated DNA repair pathways leading to senescence only in the ^KIBRAF cells. Moreover, dasatinib-induced senescence was dependent on Chk1 and p21, proteins known to mediate DNA damage-induced senescence. Dasatinib also led to a marked decrease in TAZ but not YAP protein levels. Overexpression of TAZ inhibited dasatinib-induced senescence. To investigate other vulnerabilities in ^KIBRAF NSCLC cells, we compared the sensitivity of these cells with that of ^WTBRAF NSCLC cells to 79 drugs and identified a pattern of sensitivity to EGFR and MEK inhibitors in the ^KIBRAF cells. Clinically approved EGFR and MEK inhibitors, which are better tolerated than dasatinib, could be used to treat ^KIBRAF NSCLC. Our novel finding that dasatinib induced DNA damage and subsequently activated DNA repair pathways leading to senescence in ^KIBRAF NSCLC cells represents a unique vulnerability with potential clinical applications.     

Upregulated stromal EGFR and vascular remodeling in mouse xenograft models of angiogenesis inhibitor-resistant human lung adenocarcinoma

Angiogenesis is critical for tumor growth and metastasis, and several inhibitors of angiogenesis are currently in clinical use for the treatment of cancer. However, not all patients benefit from antiangiogenic therapy, and those tumors that initially respond to treatment ultimately become resistant. The mechanisms underlying this, and the relative contributions of tumor cells and stroma to resistance, are not completely understood. Here, using species-specific profiling of mouse xenograft models of human lung adenocarcinoma, we have shown that gene expression changes associated with acquired resistance to the VEGF inhibitor bevacizumab occurred predominantly in stromal and not tumor cells. In particular, components of the EGFR and FGFR pathways were upregulated in stroma, but not in tumor cells. Increased activated EGFR was detected on pericytes of xenografts that acquired resistance and on endothelium of tumors with relative primary resistance. Acquired resistance was associated with a pattern of pericyte-covered, normalized revascularization, whereas tortuous, uncovered vessels were observed in relative primary resistance. Importantly, dual targeting of the VEGF and EGFR pathways reduced pericyte coverage and increased progression-free survival. These findings demonstrated that alterations in tumor stromal pathways, including the EGFR and FGFR pathways, are associated with, and may contribute to, resistance to VEGF inhibitors and that targeting these pathways may improve therapeutic efficacy. Understanding stromal signaling may be critical for developing biomarkers for angiogenesis inhibitors and improving combination regimens.     

Development of Kras mutant lung adenocarcinoma in mice with knockout of the airway lineage‐specific gene Gprc5a

Despite the urgency for prevention and treatment of lung adenocarcinoma (LUAD), we still do not know drivers in pathogenesis of the disease. Earlier work revealed that mice with knockout of the G‐protein coupled receptor Gprc5a develop late onset lung tumors including LUADs. Here, we sought to further probe the impact of Gprc5a expression on LUAD pathogenesis. We first surveyed GPRC5A expression in human tissues and found that GPRC5A was markedly elevated in human normal lung relative to other normal tissues and was consistently downregulated in LUADs. In sharp contrast to wild‐type littermates, Gprc5a^–/– mice treated chronically with the nicotine‐specific carcinogen NNK developed LUADs by 6 months following NNK exposure. Immunofluorescence analysis revealed that the LUADs exhibited abundant expression of surfactant protein C and lacked the clara cell marker Ccsp, suggesting that these LUADs originated from alveolar type II cells. Next, we sought to survey genome‐wide alterations in the pathogenesis of Gprc5a^–/– LUADs. Using whole exome sequencing, we found that carcinogen‐induced LUADs exhibited markedly higher somatic mutation burdens relative to spontaneous tumors. All LUADs were found to harbor somatic mutations in the Kras oncogene (p. G12D or p. Q61R). In contrast to spontaneous lesions, carcinogen‐induced Gprc5a^–/– LUADs exhibited mutations (variants and copy number gains) in additional drivers (Atm, Kmt2d, Nf1, Trp53, Met, Ezh2). Our study underscores genomic alterations that represent early events in the development of Kras mutant LUAD following Gprc5a loss and tobacco carcinogen exposure and that may constitute targets for prevention and early treatment of this disease.     

Induction of endoplasmic reticulum stress by the pro-apoptotic retinoid N-(4-hydroxyphenyl)retinamide via a reactive oxygen species-dependent mechanism in human head and neck cancer cells

N-(4-hydroxyphenyl)retinamide (4HPR), which has shown efficacy in cancer chemopreventionand therapy, induces the mitochondrial apoptosis pathway via increased generation of reactive oxygen species (ROS). ROS is also known to be able to induce an endoplasmic reticulum (ER) stress response, which can contribute to apoptosis but may also antagonize it. Therefore, we used human head and neck squamous cell carcinoma (HNSCC) cells to determine whether 4HPR affects ER stress. Different experimental approaches have indicated that 4HPR induces ER stress response: electron microscopy, which showed extensive ER dilation; splicing of the X-box binding protein 1 (XBP-1), a marker of unfolded protein response (UPR) activation; and quantitative real-time PCR and immunoblotting, which revealed the upregulation of several ER-stress associated mRNAs and proteins, including the chaperone heat shock protein HSPA1A. Most of these effects of 4HPR were abrogated by cotreatment of cells with the antioxidant 3-tert-butyl-4-hydroxyanisole (BHA) indicating that they were downstream of the increase in ROS. Furthermore, siRNA-mediated silencing and chemical inhibition of HSPA1A, which exerts either pro- or anti-apoptotic effects, decreased 4HPR-induced apoptosis. These results demonstrate that 4HPR induces ER stress and uncovered a pro-apoptotic role for HSPA1A in 4HPR-induced apoptosis.