RNASEH2B loss and PARP inhibition in advanced prostate cancer.

BACKGROUND: Clinical trials have suggested antitumor activity from PARP inhibition beyond homologous recombination deficiency (HRD). RNASEH2B loss is unrelated to HRD and preclinically sensitizes to PARP inhibition. The current study reports on RNASEH2B protein loss in advanced prostate cancer and its association with RB1 protein loss, clinical outcome and clonal dynamics during treatment with PARP inhibition in a prospective clinical trial. METHODS: Whole tumor biopsies from multiple cohorts of patients with advanced prostate cancer were interrogated using whole-exome sequencing (WES), RNA sequencing (bulk and single nucleus) and immunohistochemistry (IHC) for RNASEH2B and RB1. Biopsies from patients treated with olaparib in the TOPARP-A and TOPARP-B clinical trials were used to evaluate RNASEH2B clonal selection during olaparib treatment. RESULTS: Shallow co-deletion of RNASEH2B and adjacent RB1, co-located at chromosome 13q14, was common, deep co-deletion infrequent, and gene loss associated with lower mRNA expression. In castration-resistant PC (CRPC) biopsies, RNASEH2B and RB1 mRNA expression correlated, but single nucleus RNA sequencing indicated discordant loss of expression. IHC studies showed that loss of the two proteins often occurred independently, arguably due to stochastic second allele loss. Pre- and post-treatment metastatic CRPC (mCRPC) biopsy studies from BRCA1/2 wildtype tumors, treated on the TOPARP phase II trial, indicated that olaparib eradicates RNASEH2B-loss tumor subclones. CONCLUSION: PARP inhibition may benefit men suffering from mCRPC by eradicating tumor subclones with RNASEH2B loss. CLINICALTRIALS: gov NCT01682772FUNDING. AstraZeneca; Cancer Research UK; Medical Research Council; Cancer Research UK; Prostate Cancer UK; Movember Foundation; Prostate Cancer Foundation.

Norcantharidin inhibits the malignant progression of cervical cancer by inducing endoplasmic reticulum stress.

The antitumor effect of norcantharidin (NCTD) has been widely reported. However, whether NCTD can inhibit cervical cancer remains unknown. In the present study, it was shown that NCTD inhibited the viability of cervical cancer cells and caused cell cycle arrest in a concentration­dependent manner. Further analysis revealed that the NCTD­induced reduction in cell viability could be reversed by the inhibitor of apoptosis z­VAD­FMK and by the inhibitor of endoplasmic reticulum (ER) stress, 4­phenylbutyric acid (4­PBA). Additionally, NCTD led to the accumulation of reactive oxygen species as well as a decrease in the mitochondrial membrane potential in cervical cancer cells, whereas 4­PBA pre­treatment attenuated these alterations. In addition, NCTD increased the expression of the apoptosis­related proteins Bip, activating transcription factor (ATF) 4 and C/EBP homologous protein in a concentration­dependent manner. Moreover, NCTD significantly increased the expression of the ER stress­related signaling molecules protein kinase R­like ER kinase, inositol­requiring enzyme 1 and ATF6, but 4­PBA abolished these effects. In vivo experiments showed that NCTD significantly inhibited the growth of subcutaneous tumors in mice. Additionally, the expression of ER stress­related molecules and apoptosis­related proteins increased significantly after NCTD treatment. In conclusion, NCTD induces apoptosis by activating ER stress and ultimately curtails the progression of cervical cancer.

MEKK2 and MEKK3 orchestrate multiple signals to regulate Hippo pathway.

The Hippo pathway is an evolutionarily conserved signaling pathway that controls organ size in animals via the regulation of cell proliferation and apoptosis. It consists of a kinase cascade, in which MST1/2 and MAP4Ks phosphorylate and activate LATS1/2, which in turn phosphorylate and inhibit YAP/TAZ activity. A variety of signals can modulate LATS1/2 kinase activity to regulate Hippo pathway. However, the full mechanistic details of kinase-mediated regulation of Hippo pathway signaling remain elusive. Here, we report that TNF activates LATS1/2 and inhibits YAP/TAZ activity through MEKK2/3. Furthermore, MEKK2/3 act in parallel to MST1/2 and MAP4Ks to regulate LATS1/2 and YAP/TAZ in response to various signals, such as serum and actin dynamics. Mechanistically, we show that MEKK2/3 interact with LATS1/2 and YAP/TAZ and phosphorylate them. In addition, Striatin-interacting phosphatase and kinase (STRIPAK) complex associates with MEKK3 via CCM2 and CCM3 to inactivate MEKK3 kinase activity. Upstream signals of Hippo pathway trigger the dissociation of MEKK3 from STRIPAK complex to release MEKK3 activity. Our work has uncovered a previous unrecognized regulation of Hippo pathway via MEKK2/3 and provides new insights into molecular mechanisms for the interplay between Hippo-YAP and NF-κB signaling and the pathogenesis of cerebral cavernous malformations.

Loss of VGLL4 suppresses tumor PD-L1 expression and immune evasion.

Targeting immune checkpoints, such as PD-L1 and its receptor PD-1, has opened a new avenue for treating cancers. Understanding the regulatory mechanism of PD-L1 and PD-1 will improve the clinical response rate and efficacy of PD-1/PD-L1 blockade in cancer patients and the development of combinatorial strategies. VGLL4 inhibits YAP-induced cell proliferation and tumorigenesis through competition with YAP for binding to TEADs. However, whether VGLL4 has a role in anti-tumor immunity is largely unknown. Here, we found that disruption of Vgll4 results in potent T cell-mediated tumor regression in murine syngeneic models. VGLL4 deficiency reduces PD-L1 expression in tumor cells. VGLL4 interacts with IRF2BP2 and promotes its protein stability through inhibiting proteasome-mediated protein degradation. Loss of IRF2BP2 results in persistent binding of IRF2, a transcriptional repressor, to PD-L1 promoter. In addition, YAP inhibits IFNγ-inducible PD-L1 expression partially through suppressing the expression of VGLL4 and IRF1 by YAP target gene miR-130a. Our study identifies VGLL4 as an important regulator of PD-L1 expression and highlights a central role of VGLL4 and YAP in the regulation of tumor immunity.

Reciprocal inhibition of YAP/TAZ and NF-κB regulates osteoarthritic cartilage degradation.

Osteoarthritis is one of the leading causes of pain and disability in the aged population due to articular cartilage damage. This warrants investigation of signaling mechanisms that could protect cartilage from degeneration and degradation. Here we show in a murine model of experimental osteoarthritis that YAP activation by transgenic overexpression or by deletion of its upstream inhibitory kinases Mst1/2 preserves articular cartilage integrity, whereas deletion of YAP in chondrocytes promotes cartilage disruption. Our work shows that YAP is both necessary and sufficient for the maintenance of cartilage homeostasis in osteoarthritis. Mechanistically, inflammatory cytokines, such as TNFα or IL-1ß, trigger YAP/TAZ degradation through TAK1-mediated phosphorylation. Furthermore, YAP directly interacts with TAK1 and attenuates NF-κB signaling by inhibiting substrate accessibility of TAK1. Our study establishes a reciprocal antagonism between Hippo-YAP/TAZ and NF-κB signaling in regulating the induction of matrix-degrading enzyme expression and cartilage degradation during osteoarthritis pathogenesis.

MEKK2 and MEKK3 suppress Hedgehog pathway-dependent medulloblastoma by inhibiting GLI1 function.

Hedgehog (Hh) pathway plays a pivotal role in diverse aspects of development and postnatal physiology. Perturbation of Hh signaling and activation of GLI1 (glioma-associated oncogene 1), a dedicated transcription factor for Hh pathway, are highly associated with several cancers, such as medulloblastoma and basal cell carcinoma. Dynamic and precise control of GLI1 activity is thus important to ensure proper homeostasis and tumorigenesis. Here we show that MEKK2 (MAP3K2) and MEKK3 (MAP3K3) inhibit GLI1 transcriptional activity and oncogenic function through phosphorylation on multiple Ser/Thr sites of GLI1, which reduces GLI1 protein stability, DNA-binding ability, and increases the association of GLI1 with SUFU. Interestingly, MEKK2 and MEKK3 are responsible for FGF2-mediated inhibition on Hh signaling. Moreover, expression of MEKK2 and MEKK3 inhibits medulloblastoma cell proliferation and negatively correlates with Hh pathway activity in medulloblastoma clinical samples. Together, these findings reveal a novel noncanonical GLI1 regulation and provide a potential therapeutic target for the treatment of cancers with aberrant Hh pathway activation, such as medulloblastoma.

Hepatic loss of Lissencephaly 1 (Lis1) induces fatty liver and accelerates liver tumorigenesis in mice.

The liver is a major organ in lipid metabolism, and its malfunction leads to various diseases. Nonalcoholic fatty liver disease, the most common chronic liver disorder in developed countries, is characterized by the abnormal retention of excess lipid within hepatocytes and predisposes individuals to liver cancer. We previously reported that the levels of Lissencephaly 1 (LIS1, also known as PAFAH1B1) are down-regulated in human hepatocellular carcinoma. Following up on this observation, we found that genetic deletion of Lis1 in the mouse liver increases lipid accumulation and inflammation in this organ. Further analysis revealed that loss of Lis1 triggers endoplasmic reticulum (ER) stress and reduces triglyceride secretion. Attenuation of ER stress by addition of tauroursodeoxycholic acid (TUDCA) diminished lipid accumulation in the Lis1-deficient hepatocytes. Moreover, the Golgi stacks were disorganized in Lis1-deficient liver cells. Of note, the Lis1 liver-knockout mice exhibited increased hepatocyte ploidy and accelerated development of liver cancer after exposure to the liver carcinogen diethylnitrosamine (DEN). Taken together, these findings suggest that reduced Lis1 levels can spur the development of liver diseases from steatosis to liver cancer and provide a useful model for delineating the molecular pathways that lead to these diseases.