Targeting PELP1 Attenuates Angiogenesis and Enhances Chemotherapy Efficiency in Colorectal Cancer

Abnormal angiogenesis is one of the important hallmarks of colorectal cancer as well as other solid tumors. Optimally, anti-angiogenesis therapy could restrain malignant angiogenesis to control tumor expansion. PELP1 is as a scaffolding oncogenic protein in a variety of cancer types, but its involvement in angiogenesis is unknown. In this study, PELP1 was found to be abnormally upregulated and highly coincidental with increased MVD in CRC. Further, treatment with conditioned medium (CM) from PELP1 knockdown CRC cells remarkably arrested the function of human umbilical vein endothelial cells (HUVECs) compared to those treated with CM from wildtype cells. Mechanistically, the STAT3/VEGFA axis was found to mediate PELP1-induced angiogenetic phenotypes of HUVECs. Moreover, suppression of PELP1 reduced tumor growth and angiogenesis in vivo accompanied by inactivation of STAT3/VEGFA pathway. Notably, in vivo, PELP1 suppression could enhance the efficacy of chemotherapy, which is caused by the normalization of vessels. Collectively, our findings provide a preclinical proof of concept that targeting PELP1 to decrease STAT3/VEGFA-mediated angiogenesis and improve responses to chemotherapy due to normalization of vessels. Given the newly defined contribution to angiogenesis of PELP1, targeting PELP1 may be a potentially ideal therapeutic strategy for CRC as well as other solid tumors.

RPRD1A stabilizes NRF2 and aggravates HCC progression through competing with p62 for TRIM21 binding

NRF2 is the master transcriptional activator of cytoprotective genes and Kelch-like ECH-associated protein 1 (Keap1), a biosensor for electrophiles and oxidation, promotes NRF2 degradation in unstressed conditions. SQSTM1/p62, an oncogenic protein aberrantly accumulated in hepatocellular carcinoma (HCC), binds and sequestrates Keap1, leading to the prevention of NRF2 degradation. Here, we show that p15INK4b-related sequence/regulation of nuclear pre-mRNA domain-containing protein 1A (RPRD1A) is highly expressed in HCC tumors and correlated with aggressive clinicopathological features. RPRD1A competitively interacts with TRIM21, an E3 ubiquitin ligase of p62, resulting in the decrease of p62 ubiquitination and the increased sequestration for Keap1. Therefore, RPRD1A enhances the nuclear translocation of NRF2, which induces gene expression for counteracting oxidative stress, maintaining cancer cells survival, and promoting HCC development. Moreover, disturbing the redox homeostasis of cancer cells by genetic knockdown of RPRD1A sensitizes cancer cells to platinum-induced cell death. Our study reveals RPRD1A is involved in the oxidative stress defense program and highlights the therapeutic benefits of targeting pathways that support antioxidation.

HIF1 and ID1 Interplay Confers Adaptive Survival to HIF1α-Inhibition

Hypoxia is a universal pathological feature of solid tumors. Hypoxic tumor cells acquire metastatic and lethal phenotypes primarily through the activities of hypoxia-inducible factor 1 alpha (HIF1α). Therefore, HIF1α is considered as a promising therapeutic target. However, HIF inhibitors have not proven to be effective in clinical testing. The underlying mechanism is unclear. We report that oncogenic protein ID1 is upregulated in hypoxia by HIF1α shRNA or pharmacological inhibitors. In turn, ID1 supports tumor growth in hypoxia in vitro and in xenografts in vivo, conferring adaptive survival response and resistance. Mechanistically, ID1 proteins interfere HIF1-mediated gene transcription activation, thus ID1 protein degradation is accelerated by HIF1α-dependent mechanisms in hypoxia. Inhibitions of HIF1α rescues ID1, which compensates the loss of HIF1α by the upregulation of GLS2 and glutamine metabolism, thereby switching the metabolic dependency of HIF1α -inhibited cells from glucose to glutamine.

Targeting DDX3X Helicase Activity with BA103 Shows Promising Therapeutic Effects in Preclinical Glioblastoma Models

DDX3X is an ATP-dependent RNA helicase that has recently attracted interest for its involvement in viral replication and oncogenic progression. Starting from hit compounds previously identified by our group, we have designed and synthesized a new series of DDX3X inhibitors that effectively blocked its helicase activity. These new compounds were able to inhibit the proliferation of cell lines from different cancer types, also in DDX3X low-expressing cancer cell lines. According to the absorption, distribution, metabolism, elimination properties, and antitumoral activity, compound BA103 was chosen to be further investigated in glioblastoma models. BA103 determined a significant reduction in the proliferation and migration of U87 and U251 cells, downregulating the oncogenic protein β-catenin. An in vivo evaluation demonstrated that BA103 was able to reach the brain and reduce the tumor growth in xenograft and orthotopic models without evident side effects. This study represents the first demonstration that DDX3X-targeted small molecules are feasible and promising drugs also in glioblastoma.

Molecular Insights Into the Interaction of HPV-16 E6 Variants Against MAGI-1 PDZ1 Domain

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein-protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

PMEPA1/TMEPAI Is a Unique Tumorigenic Activator of AKT Promoting Proteasomal Degradation of PHLPP1 in Triple-Negative Breast Cancer Cells

Transmembrane prostate androgen-induced protein (TMEPAI), also known as PMEPA1, is highly expressed in many types of cancer and promotes oncogenic abilities. However, the mechanisms whereby TMEPAI facilitates tumorigenesis are not fully understood. We previously established TMEPAI-knockout (KO) cells from human triple-negative breast cancer (TNBC) cell lines and found that TMEPAI-KO cells showed reduced tumorigenic abilities. Here, we report that TMEPAI-KO cells upregulated the expression of pleckstrin homology (PH) domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) and suppressed AKT Ser473 phosphorylation, which was consistent with TCGA dataset analysis. Additionally, the knockdown (KD) of PHLPP1 in TMEPAI-KO cells partially but significantly rescued AKT Ser473 phosphorylation, as well as in vitro and in vivo tumorigenic activities, thus showing that TMEPAI functions as an oncogenic protein through the regulation of PHLPP1 subsequent to AKT activation. Furthermore, we demonstrated that TMEPAI PPxY (PY) motifs are essential for binding to NEDD4-2, an E3 ubiquitin ligase, and PHLPP1-downregulatory ability. Moreover, TMEPAI enhanced the complex formation of PHLPP1 with NEDD4-2 and PHLPP1 polyubiquitination, which leads to its proteasomal degradation. These findings indicate that the PY motifs of TMEPAI suppress the amount of PHLPP1 and maintain AKT Ser473 phosphorylation at high levels to enhance the tumorigenic potentiality of TNBC.

USP7 inhibition induces apoptosis in glioblastoma by enhancing ubiquitination of ARF4

Background Glioblastomas (GBMs) are grade IV central nervous system tumors characterized by a poor prognosis and a short median overall survival. Effective induction of GBM cell death is difficult because the GBM cell population is genetically unstable, resistant to chemotherapy and highly angiogenic. In recent studies, ubiquitin-specific protease 7 (USP7) is shown to scavenge ubiquitin from oncogenic protein substrates, so effective inhibition of USP7 may be a potential key treatment for GBM. Methods Immunohistochemistry and western blotting were used to detect the expression of USP7 in GBM tissues. In vitro apoptosis assay of USP7 inhibition was performed by western blotting, immunofluorescence, and flow cytometry. Anti-apoptotic substrates of USP7 were defined by Co-IP and TMT proteomics. Western blotting and IP were used to verify the relationship between USP7 and its substrate. In an in vivo experiment using an intracranial xenograft model in nude mice was constructed to assess the therapeutic effect of target USP7. Results Immunohistochemistry and western blotting confirmed that USP7 was significantly upregulated in glioblastoma samples. In in vitro experiments, inhibition of USP7 in GBM induced significant apoptosis. Co-IP and TMT proteomics identified a key anti-apoptotic substrate of USP7, ADP-ribosylation factor 4 (ARF4). Western blotting and IP confirmed that USP7 interacted directly with ARF4 and catalyzed the removal of the K48-linked polyubiquitinated chain that binded to ARF4. In addition, in vivo experiments revealed that USP7 inhibition significantly suppressed tumor growth and promoted the expression of apoptotic genes. Conclusions Targeted inhibition of USP7 enhances the ubiquitination of ARF4 and ultimately mediates the apoptosis of GBM cells. In a clinical sense, P5091 as a novel specific inhibitor of USP7 may be an effective approach for the treatment of GBM.

Insights in the Interaction of HPV-16 E6 and Its Variants (E-G350, E-A176/G350, E-C188/G350, AAa and AAc) with MAGI-1

Oncogenic protein E6 from Human Papilloma Virus 16 (HPV-16) mediates the degradation of Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1), throughout the interaction of its protein binding motif (PBM) with the Discs-large homologous regions 1 (PDZ1) domain of MAG1-1. Generic variation in the E6 gene that translates to changes in the protein’s amino acidic sequence modifies the interaction of E6 with the cellular protein MAGI-1. MAGI-1 is a scaffolding protein found at tight junctions of epithelial cells, where it interacts with a variety of proteins regulating signaling pathways. MAGI-1 is a multidomain protein containing two WW (rsp-domain-9), one guanylate kinase-like, and six PDZ domains. PDZ domains played an important role in the function of MAGI-1 and served as targets for several viral proteins including the HPV-16 E6. The aim of this work was to evaluate, with an in silico approach, employing molecular dynamics simulation and protein-protein docking, the interaction of the intragenic variants E-G350 (L83V), E-C188/G350 (E29Q/L83V), E-A176/G350 (D25N/L83V), E6-AAa (Q14H/H78Y/83V) y E6-AAc (Q14H/I27RH78Y/L83V) and E6-reference of HPV-16 with MAGI-1. We found that variants E-G350, E-C188/G350, E-A176/G350, AAa and AAc increase their affinity to our two models of MAGI-1 compared to E6-reference.

YBX1 mediates translation of oncogenic transcripts to control cell competition in AML

Persistence of malignant clones is a major determinant of adverse outcome in patients with hematologic malignancies. Despite the fact that the majority of patients with acute myeloid leukemia (AML) achieve complete remission after chemotherapy, a large proportion of them relapse as a result of residual malignant cells. These persistent clones have a competitive advantage and can re-establish disease. Therefore, targeting strategies that specifically diminish cell competition of malignant cells while leaving normal cells unaffected are clearly warranted. Recently, our group identified YBX1 as a mediator of disease persistence in JAK2-mutated myeloproliferative neoplasms. The role of YBX1 in AML, however, remained so far elusive. Here, inactivation of YBX1 confirms its role as an essential driver of leukemia development and maintenance. We identify its ability to amplify the translation of oncogenic transcripts, including MYC, by recruitment to polysomal chains. Genetic inactivation of YBX1 disrupts this regulatory circuit and displaces oncogenic drivers from polysomes, with subsequent depletion of protein levels. As a consequence, leukemia cells show reduced proliferation and are out-competed in vitro and in vivo, while normal cells remain largely unaffected. Collectively, these data establish YBX1 as a specific dependency and therapeutic target in AML that is essential for oncogenic protein expression.