Necrocide 1 mediates necrotic cell death and immunogenic response in human cancer cells.

Many anticancer agents induce apoptosis, mitotic catastrophe or cellular senescence. Here, we report the functional characterization of an experimental inducer of tumor necrosis factor (TNF)-independent necrosis, necrocide-1 (NC1). NC1 (but not its stereoisomer) killed a panel of human cancer cells (but not normal cells) at nanomolar concentrations and with a non-apoptotic, necrotic morphotype, both in vitro and in vivo. NC1-induced killing was not inhibited by caspase blockers, anti-apoptotic BCL2 overexpression or TNFα neutralization, suggesting that NC1 elicits a bona fide necrotic pathway. However, pharmacological or genetic inhibition of necroptosis, pyroptosis and ferroptosis failed to block NC1-mediated cell death. Instead, NC1 elicited reactive oxygen species (ROS) production by mitochondria, and elimination of mitochondrial DNA, quenching of mitochondrial ROS, as well as blockade of mitochondrial permeability transition with cyclosporine A, interfered with NC1-induced cell death. NC1 induced hallmarks of immunogenic cell death incurring calreticulin (CALR) exposure, ATP secretion and high mobility group box 1 (HMGB1) release. Taken together, these data identify a previously uncharacterized signaling cascade leading to an immunogenic variant of mitochondrion-regulated necrosis, supporting the notion that eliciting regulated necrosis may constitute a valid approach for anticancer therapy.

Ubiquitin ligase E3 HUWE1/MULE targets transferrin receptor for degradation and suppresses ferroptosis in acute liver injury.

Hepatic ischemia followed by reperfusion (I/R), a major clinical problem during liver surgical procedures, can induce liver injury with severe cell death including ferroptosis which is characterized by iron-dependent accumulation of lipid peroxidation. The HECT domain-containing ubiquitin E3 ligase HUWE1 (also known as MULE) was initially shown to promote apoptosis. However, our preliminary study demonstrates that high expression of HUWE1 in the liver donors corelates with less injury and better hepatic function after liver transplantation in patients. Thus, we investigate the role of HUWE1 in acute liver injury, and identify HUWE1 as a negative ferroptosis modulator through transferrin receptor 1(TfR1). Deficiency of Huwe1 in mice hepatocytes (HKO) exacerbated I/R and CCl4-induced liver injury with more ferroptosis occurrence. Moreover, Suppression of Huwe1 remarkably enhances cellular sensitivity to ferroptosis in primary hepatocytes and mouse embryonic fibroblasts. Mechanistically, HUWE1 specifically targets TfR1 for ubiquitination and proteasomal degradation, thereby regulates iron metabolism. Importantly, chemical and genetic inhibition of TfR1 dramatically diminishes the ferroptotic cell death in Huwe1 KO cells and Huwe1 HKO mice. Therefore, HUWE1 is a potential protective factor to antagonize both aberrant iron accumulation and ferroptosis thereby mitigating acute liver injury. These findings may provide clinical implications for patients with the high-expression Huwe1 alleles.

Disrupted intraflagellar transport due to IFT74 variants causes Joubert syndrome.

PURPOSE: Ciliopathies are a group of disorders caused by defects of the cilia. Joubert syndrome (JBTS) is a recessive and pleiotropic ciliopathy that causes cerebellar vermis hypoplasia and psychomotor delay. Although the intraflagellar transport (IFT) complex serves as a key module to maintain the ciliary structure and regulate ciliary signaling, the function of IFT in JBTS remains largely unknown. We aimed to explore the impact of IFT dysfunction in JBTS. METHODS: Exome sequencing was performed to screen for pathogenic variants in IFT genes in a JBTS cohort. Animal model and patient-derived fibroblasts were used to evaluate the pathogenic effects of the variants. RESULTS: We identified IFT74 as a JBTS-associated gene in three unrelated families. All the affected individuals carried truncated variants and shared one missense variant (p.Q179E) found only in East Asians. The expression of the human p.Q179E-IFT74 variant displayed compromised rescue effects in zebrafish ift74 morphants. Attenuated ciliogenesis; altered distribution of IFT proteins and ciliary membrane proteins, including ARL13B, INPP5E, and GPR161; and disrupted hedgehog signaling were observed in patient fibroblasts with IFT74 variants. CONCLUSION: IFT74 is identified as a JBTS-related gene. Cellular and biochemical mechanisms are also provided.

KLF4K409Q-mutated meningiomas show enhanced hypoxia signaling and respond to mTORC1 inhibitor treatment.

Meningioma represents the most common primary brain tumor in adults. Recently several non-NF2 mutations in meningioma have been identified and correlated with certain pathological subtypes, locations and clinical observations. Alterations of cellular pathways due to these mutations, however, have largely remained elusive. Here we report that the Krueppel like factor 4 (KLF4)-K409Q mutation in skull base meningiomas triggers a distinct tumor phenotype. Transcriptomic analysis of 17 meningioma samples revealed that KLF4K409Q mutated tumors harbor an upregulation of hypoxia dependent pathways. Detailed in vitro investigation further showed that the KLF4K409Q mutation induces HIF-1α through the reduction of prolyl hydroxylase activity and causes an upregulation of downstream HIF-1α targets. Finally, we demonstrate that KLF4K409Q mutated tumors are susceptible to mTOR inhibition by Temsirolimus. Taken together, our data link the KLF4K409Q mediated upregulation of HIF pathways to the clinical and biological characteristics of these skull base meningiomas possibly opening new therapeutic avenues for this distinct meningioma subtype.

PHD3 Controls Lung Cancer Metastasis and Resistance to EGFR Inhibitors through TGFα.

Lung cancer is the leading cause of cancer-related death worldwide, in large part due to its high propensity to metastasize and to develop therapy resistance. Adaptive responses to hypoxia and epithelial-mesenchymal transition (EMT) are linked to tumor metastasis and drug resistance, but little is known about how oxygen sensing and EMT intersect to control these hallmarks of cancer. Here, we show that the oxygen sensor PHD3 links hypoxic signaling and EMT regulation in the lung tumor microenvironment. PHD3 was repressed by signals that induce EMT and acted as a negative regulator of EMT, metastasis, and therapeutic resistance. PHD3 depletion in tumors, which can be caused by the EMT inducer TGFß or by promoter methylation, enhanced EMT and spontaneous metastasis via HIF-dependent upregulation of the EGFR ligand TGFα. In turn, TGFα stimulated EGFR, which potentiated SMAD signaling, reinforcing EMT and metastasis. In clinical specimens of lung cancer, reduced PHD3 expression was linked to poor prognosis and to therapeutic resistance against EGFR inhibitors such as erlotinib. Reexpression of PHD3 in lung cancer cells suppressed EMT and metastasis and restored sensitivity to erlotinib. Taken together, our results establish a key function for PHD3 in metastasis and drug resistance and suggest opportunities to improve patient treatment by interfering with the feedforward signaling mechanisms activated by PHD3 silencing.Significance: This study links the oxygen sensor PHD3 to metastasis and drug resistance in cancer, with implications for therapeutic improvement by targeting this system. Cancer Res; 78(7); 1805-19. ©2018 AACR.

Lung Tumor Suppressor GPRC5A Binds EGFR and Restrains Its Effector Signaling.

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a(-/-) mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a(-/-) mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a(-/-) MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a(-/-) mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non-small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801-14. ©2015 AACR.

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway.

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1ß and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.

Sumoylation of hypoxia inducible factor-1α and its significance in cancer.

Hypoxia-inducible factor-1 (HIF-1) is a key heterodimeric transcription factor for the cellular adaptive response to hypoxia, a common feature of the microenvironment in solid tumors. The transcriptional activity, protein stabilization, protein-protein interactions and cellular localization of HIF-1α, an oxygen-sensitive subunit of HIF-1, are mainly modulated by various post-translational modifications. Recently, we reported that polycomb chromobox 4 (Cbx4) governs the transcriptional activity of HIF-1α by enhancing its sumoylation at K391 and K477, through which Cbx4 potentiates angiogenesis of hepatocellular carcinoma. This review summarizes the current knowledge of HIF-1α sumoylation and its roles in the pathogenesis of cancer.

Polycomb chromobox 4 enhances migration and pulmonary metastasis of hepatocellular carcinoma cell line MHCC97L.

We recently report that the expression of polycomb chromobox 4 (Cbx4) is significantly correlated with the overall survival of a great cohort of hepatocellular carcinoma (HCC) patients and it enhances hypoxia-induced vascular endothelial growth factor (VEGF) expression and angiogenesis in HCC cells through enhancing sumoylation of hypoxia inducible factor-1alpha (HIF-1α). Here we continue to investigate the potential effects of Cbx4 on the migration and metastasis of the metastatic HCC cell line MHCC97L. Our results show that Cbx4 overexpression in the cell line increases the in vitro vessel formation of vascular endothelial cells in its SUMO interaction motifs-dependent manner, and promotes the in vitro migration of the cancer cell, which can be effectively abrogated by anti-VEGF antibody. Although Cbx4 expression does not impact the in vitro growth of MHCC97L cells, it still promotes the progression and metastasis of orthotopically transplanted tumors in nude mice. These results further support the role of Cbx4 as a SUMO E3 ligase in the progression and metastasis of HCC.

Cbx4 governs HIF-1α to potentiate angiogenesis of hepatocellular carcinoma by its SUMO E3 ligase activity.

Cbx4 is a polycomb group protein that is also a SUMO E3 ligase, but its potential roles in tumorigenesis remain to be explored. Here, we report that Cbx4, but not other members of the Cbx family, enhances hypoxia-induced vascular endothelial growth factor (VEGF) expression and angiogenesis in hepatocellular carcinoma (HCC) cells through enhancing HIF-1α sumoylations at K391 and K477 in its two SUMO-interacting motifs-dependent mechanisms and increasing transcriptional activity of HIF-1. The Cbx4 expression is significantly correlated with VEGF expression, angiogenesis, and the overall survival of HCC patients and also in subcutaneously and orthotopically transplanted mice HCC models. Collectively, our findings demonstrate that Cbx4 plays a critical role in tumor angiogenesis by governing HIF-1α protein.