INTRODUCTION: The efficacy of chemotherapeutic agents in killing cancer cells is mainly attributed to the induction of apoptosis. However, the tremendous efforts on enhancing apoptosis-related mechanisms have only moderately improved lung cancer chemotherapy, suggesting that other cell death mechanisms such as necroptosis could be involved. In this study, we investigated the role of the necroptosis pathway in the responsiveness of nonsmall cell lung cancer (NSCLC) to chemotherapy. METHODS: In vitro cell culture and in vivo xenograft tumor therapy models and clinical sample studies are combined in studying the role of necroptosis in chemotherapy and mechanism of necroptosis suppression involving RIP3 expression regulation. RESULTS: While chemotherapeutic drugs were able to induce necroptotic cell death, this pathway was suppressed in lung cancer cells at least partly through downregulation of RIP3 expression. Ectopic RIP3 expression significantly sensitized lung cancer cells to the cytotoxicity of anticancer drugs such as cisplatin, etoposide, vincristine, and adriamycin. In addition, RIP3 suppression was associated with RIP3 promoter methylation, and demethylation partly restored RIP3 expression and increased chemotherapeutic-induced necroptotic cell death. In a xenograft tumor therapy model, ectopic RIP3 expression significantly sensitized anticancer activity of cisplatin in vivo. Furthermore, lower RIP3 expression was associated with worse chemotherapy response in NSCLC patients. CONCLUSION: Our results indicate that the necroptosis pathway is suppressed in lung cancer through RIP3 promoter methylation, and reactivating this pathway should be exploited for improving lung cancer chemotherapy.

Platinum anticancer agents are essential components in chemotherapeutic regimens for non–small‐cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum‐based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino‐sugar N‐acetyl‐glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose‐6‐phosphate amidotransferase (GFAT), the rate‐limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose‐regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol‐requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin‐induced cell apoptosis. These data identify GFAT‐mediated HBP as a target for improving platinum‐based chemotherapy for NSCLC.

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by the Chal-24 and TRAIL combination, which was associated with attenuation of c-FLIPL and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy.

Mucin 1 (MUC1) is a tumor antigen that is aberrantly overexpressed in various cancers, including lung cancer. Our previous in vitro studies showed that MUC1 facilitates carcinogen-induced EGFR activation and transformation in human lung bronchial epithelial cells (HBECs), which along with other reports suggests an oncogenic property for MUC1 in lung cancer. However, direct evidence for the role of MUC1 in lung carcinogenesis is lacking. In this study, we used the 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced A/J mouse lung tumor model to investigate the effect of whole-body Muc1 knockout (KO) on carcinogen-induced lung carcinogenesis. Surprisingly, lung tumor multiplicity was significantly increased in Muc1 KO compared to wild-type (WT) mice. The EGFR/AKT pathway was unexpectedly activated, and expression of the EGFR ligand epiregulin (EREG) was increased in the lung tissues of the Muc1 KO compared to the WT mice. EREG stimulated proliferation and protected against cigarette smoke extract (CSE)-induced cytotoxicity in in vitro cultured human bronchial epithelial cells. Additionally, we determined that MUC1 was expressed in human fibroblast cell lines where it suppressed CSE-induced EREG production. Further, suppression of MUC1 cellular activity with GO-201 enhanced EREG production in lung cancer cells, which in turn protected cancer cells from GO-201-induced cell death. Moreover, an inverse association between MUC1 and EREG was detected in human lung cancer, and EREG expression was inversely associated with patient survival. Together, these results support a promiscuous role of MUC1 in lung cancer development that may be related to cell-type specific functions of MUC1 in the tumor microenvironment, and MUC1 deficiency in fibroblasts and malignant cells results in increased EREG production that activates the EGFR pathway for lung carcinogenesis.

Immunotherapy using checkpoint blockers (antibodies) has been a major advance in recent years in the management of various types of solid cancers including lung cancer. One target of checkpoint blockers is programmed death ligand 1 (PD-L1) expressed by cancer cells, which engages programmed death 1 (PD-1) on T cells and Natural Killer (NK) cells resulting in suppression of their activation and cancer-killing function, respectively. Apart from antibodies, other clinically relevant agents that can inhibit PD-L1 are limited. PD-L1 protein stability depends on its glycosylation. Here we show that L-glutamine:D-fructose amidotransferase 1 (GFAT1) a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) which produces uridine diphosphate-N-acetyl-β-glucosamine (UDP-GlcNAc), a precursor for glycosylation, is required for the stability of PD-L1 protein. Inhibition of GFAT1 activity markedly reduced interferon γ (IFNγ)-induced PD-L1 levels in various lung cancer cell lines. GFAT1 inhibition suppressed glycosylation of PD-L1 and accelerated its proteasomal degradation. Importantly, inhibition of GFAT1 in IFNγ-treated cancer cells enhanced the activation of T cells and the cancer-killing activity of NK cells. These findings support using GFAT1 inhibitors to manipulate PD-L1 protein level that could augment the efficacy of immunotherapy for lung cancer.

Combination chemotherapy is an effective strategy for increasing anticancer efficacy, reducing side effects and alleviating drug resistance. Here we report that combination of the recently identified novel chalcone derivative, chalcone-24 (Chal-24), and TNF-related apoptosis-inducing ligand (TRAIL) significantly increases cytotoxicity in lung cancer cells. Chal-24 treatment significantly enhanced TRAIL-induced activation of caspase-8 and caspase-3, and the cytotoxicity induced by combination of these agents was effectively suppressed by the pan-caspase inhibitor z-VAD-fmk. Chal-24 and TRAIL combination suppressed expression of cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein large (c-FLIPL) and cellular inhibitor of apoptosis proteins (c-IAPs), and ectopic expression of c-FLIPL and c-IAPs inhibited the potentiated cytotoxicity. In addition, TRAIL and Chal-24 cooperatively activated autophagy. Suppression of autophagy effectively attenuated cytotoxicity induced by the combination of Chal-24 and TRAIL, which was associated with attenuation of c-FLIPL and c-IAPs degradation. Altogether, these results suggest that Chal-24 potentiates the anticancer activity of TRAIL through autophagy-mediated degradation of c-FLIPL and c-IAPs, and that combination of Chal-24 and TRAIL could be an effective approach in improving chemotherapy efficacy. Citation Format: Xiuling Xu, Jennings Xu, Shaoqing Shi, Qiong Wang, Bryanna Saxton, Chengguo Xing, Yong Lin. Combination of TRAIL and Chal-24 synergistically induces autophagy-mediated apoptosis in lung cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A06.

Immunotherapy using checkpoint blockers (antibodies) has been a major advance in recent years in the management of various types of solid cancers including lung cancer. One target of checkpoint blockers is programmed death ligand 1 (PD-L1) expressed by cancer cells, which engages programmed death 1 (PD-1) on T cells and Natural Killer (NK) cells resulting in suppression of their activation and cancer-killing function, respectively. Apart from antibodies, other clinically relevant agents that can inhibit PD-L1 are limited. PD-L1 protein stability depends on its glycosylation. Here we show that L-glutamine:D-fructose amidotransferase 1 (GFAT1) a rate-limiting enzyme of the hexosamine biosynthesis pathway (HBP) which produces uridine diphosphate-N-acetyl-β-glucosamine (UDP-GlcNAc), a precursor for glycosylation, is required for the stability of PD-L1 protein. Inhibition of GFAT1 activity markedly reduced interferon γ (IFNγ)-induced PD-L1 levels in various lung cancer cell lines. GFAT1 inhibition suppressed glycosylation of PD-L1 and accelerated its proteasomal degradation. Importantly, inhibition of GFAT1 in IFNγ-treated cancer cells enhanced the activation of T cells and the cancer-killing activity of NK cells. These findings support using GFAT1 inhibitors to manipulate PD-L1 protein level that could augment the efficacy of immunotherapy for lung cancer.

US homeland security concerns related to potential misuse of γ-ray-emitting radiation sources employed in radiobiological research (eg, shielded cesium-137 irradiators) led to recommendations by the National Research Council to conduct studies into possibly replacing γ-ray irradiators used in research involving small rodent and other models with X-ray instruments. A limiting factor is suitability of the X-ray photon energy spectra. The objective of our research was to demonstrate the suitability of the radiation energy spectrum of 320-kV X rays after filtration (HVL = 4 mm Cu) for in-vivo cytotoxicity studies in immunodeficient C.B-17 SCID mice. By using a previously-published Hazard Function (HF) model to characterize dose-response relationships for in vivo bone marrow and spleen cell survival fractions and also to characterize the acute lethality risk (hematopoietic syndrome mode) we demonstrate that the filtered 320-kV X-ray beam appears suitable for such studies. A key finding for C.B-17 SCID mice when compared to results previously obtained for immunocompetent C.B-17 mice is that the immunodeficient mice appear to be more radioresistant, implicating a possible role of the immune system capacity in radiosensitivity of mammals.