The p38-interacting protein p38IP suppresses TCR and LPS signaling by targeting TAK1.

Negative regulation of immunoreceptor signaling is required for preventing hyperimmune activation and maintaining immune homeostasis. The roles of p38IP in immunoreceptor signaling remain unclear. Here, we show that p38IP suppresses T-cell receptor (TCR)/LPS-activated NF-κB and p38 by targeting TAK1 kinase and that p38IP protein levels are downregulated in human PBMCs from rheumatoid arthritis (RA) patients, inversely correlating with the enhanced activity of NF-κB and p38. Mechanistically, p38IP interacts with TAK1 to disassemble the TAK1-TAB (TAK1-binding protein) complex. p38IP overexpression decreases TCR-induced binding of K63-linked polyubiquitin (polyUb) chains to TAK1 but increases that to TAB2, and p38IP knockdown shows the opposite effects, indicating unanchored K63-linked polyUb chain transfer from TAB2 to TAK1. p38IP dynamically interacts with TAK1 upon stimulation, because of the polyUb chain transfer and the higher binding affinity of TAK1 and p38IP for polyUb-bound TAB2 and TAK1, respectively. Moreover, p38IP scaffolds the deubiquitinase USP4 to deubiquitinate TAK1 once TAK1 is activated. These findings reveal a novel role and the mechanisms of p38IP in controlling TCR/LPS signaling and suggest that p38IP might participate in RA pathogenesis.

Vesicle transporter GOLT1B mediates the cell membrane localization of DVL2 and PD-L2 and promotes colorectal cancer metastasis.

BACKGROUND: Colorectal cancer (CRC) is the third most diagnosed and second leading cause of cancer death worldwide. Hallmark proteins processing is usually dysregulated in cancers. Finding key regulatory molecules is of great importance for CRC metastasis intervention. GOLT1B is a vesicle transport protein which is involved in cytosolic proteins trafficking. However, its role in cancer has never been addressed. METHODS: CRC cell lines and subcutaneous xenograft animal model were utilized to investigate the biological function of GOLT1B. Patients samples were used to validate the correlation between GOLT1B and clinical outcome. In vivo targeted delivery of GOLT1B-siRNA was investigated in PDX (Patient derived tumor xenograft) model. RESULTS: We found that GOLT1B was highly expressed in CRC, and was an independent prognostic marker of overall survival (OS) and progression free survival (PFS). GOLT1B could promote CRC metastasis in vitro and in vivo. GOLT1B overexpression could increase DVL2 level and enhance its plasma membrane translocation, which subsequently activated downstream Wnt/ß-catenin pathway and increase the nuclear ß-catenin level, hence induce epithelial-mesenchymal transition (EMT). In addition, GOLT1B could also interact with PD-L2 and increase its membrane level. Co-culture of GOLT1B-overexpresed CRC cells with Jurkat cells significantly induced T cells apoptosis, which might further promote cancer cell the migration and invasion. Further, targeted delivery of GOLT1B siRNA could significantly inhibit tumor progression in GOLT1B highly expressed PDX model. CONCLUSION: Taken together, our findings suggest that the vesicle transporter GOLT1B could promote CRC metastasis not only by assisting DVL2 translocation and activating Wnt/ß-catenin pathway, but also facilitating PD-L2 membrane localization to induce immune suppression. Targeted inhibition of GOLT1B could be a potential therapeutic strategy for CRC treatment.

Discovery of Kinetin in inhibiting colorectal cancer progression via enhancing PSMB1-mediated RAB34 degradation.

Colorectal cancer (CRC) is one of the most prevalent malignancies worldwide. Understanding the underlying mechanism driving CRC progression and identifying potential therapeutic drug targets are of utmost urgency. We previously utilized LC-MS-based proteomic profiling to identify proteins associated with postoperative progression in stage II/III CRC. Here, we revealed that proteasome subunit beta type-1 (PSMB1) is an independent predictor for postoperative progression in stage II/III CRC. Mechanistically, PSMB1 binds directly to onco-protein RAB34 and promotes its proteasome-dependent degradation, potentially leading to the inactivation of the MEK/ERK signaling pathway and inhibition of CRC progression. To further identify potential anticancer drugs, we screened a library of 2509 FDA-approved drugs using computer-aided drug design (CADD) and identified Kinetin as a potentiating agent for PSMB1. Functional assays confirmed that Kinetin enhanced the interaction between PSMB1 and RAB34, hence facilitated the degradation of RAB34 protein and decreased the MEK/ERK phosphorylation. Kinetin suppresses CRC progression in patient-derived xenograft (PDX) and liver metastasis models. Conclusively, our study identifies PSMB1 as a potential biomarker and therapeutic target for CRC, and Kinetin as an anticancer drug by enhancing proteasome-dependent onco-protein degradation.

CRISPR screens reveal convergent targeting strategies against evolutionarily distinct chemoresistance in cancer.

Resistance to chemotherapy has been a major hurdle that limits therapeutic benefits for many types of cancer. Here we systematically identify genetic drivers underlying chemoresistance by performing 30 genome-scale CRISPR knockout screens for seven chemotherapeutic agents in multiple cancer cells. Chemoresistance genes vary between conditions primarily due to distinct genetic background and mechanism of action of drugs, manifesting heterogeneous and multiplexed routes towards chemoresistance. By focusing on oxaliplatin and irinotecan resistance in colorectal cancer, we unravel that evolutionarily distinct chemoresistance can share consensus vulnerabilities identified by 26 second-round CRISPR screens with druggable gene library. We further pinpoint PLK4 as a therapeutic target to overcome oxaliplatin resistance in various models via genetic ablation or pharmacological inhibition, highlighting a single-agent strategy to antagonize evolutionarily distinct chemoresistance. Our study not only provides resources and insights into the molecular basis of chemoresistance, but also proposes potential biomarkers and therapeutic strategies against such resistance.

Aquaporin 9 is involved in CRC metastasis through DVL2-dependent Wnt/ß-catenin signaling activation.

Background: Aquaporin 9 (AQP9) is permeable to water or other small molecules, and plays an important role in various cancers. We previously found that AQP9 was related to the efficacy of chemotherapy in patients with colorectal cancer (CRC). This study aimed to identify the role and regulatory mechanism of AQP9 in CRC metastasis. Methods: The clinical significance of AQP9 was analysed by using bioinformatics and tissue microarray. Transcriptome sequencing, Dual-Luciferase Reporter Assay, Biacore, and co-immunoprecipitation were employed to demonstrate the regulatory mechanism of AQP9 in CRC. The relationship between AQP9 and CRC metastasis was verified in vitro and in vivo by using real-time cell analysis assay, high content screening, and liver metastasis models of nude mice. Results: We found that AQP9 was highly expressed in metastatic CRC. AQP9 overexpression reduced cell roundness and enhanced cell motility in CRC. We further showed that AQP9 interacted with Dishevelled 2 (DVL2) via the C-terminal SVIM motif, resulting in DVL2 stabilization and the Wnt/ß-catenin pathway activation. Additionally, we identified the E3 ligase neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) as a modulator regulating the ubiquitination and degradation of AQP9. Conclusions: Collectively, our study revealed the important role of AQP9 in regulating DVL2 stabilization and Wnt/ß-catenin signaling to promote CRC metastasis. Targeting the NEDD4L-AQP9-DVL2 axis might have therapeutic usefulness in metastatic CRC treatment.

The Role of RAB GTPases and Its Potential in Predicting Immunotherapy Response and Prognosis in Colorectal Cancer.

Background: Colorectal cancer (CRC) is the third most common cancer worldwide, in which aberrant activation of the RAS signaling pathway appears frequently. RAB proteins (RABs) are the largest Ras small GTPases superfamily that regulates intracellular membrane trafficking pathways. The dysregulation of RABs have been found in various diseases including cancers. Compared with other members of Ras families, the roles of RABs in colorectal cancer are less well understood. Methods: We analyzed the differential expression and clinicopathological association of RABs in CRC using RNA sequencing and genotyping datasets from TCGA samples. Moreover, the biological function of RAB17 and RAB34 were investigated in CRC cell lines and patient samples. Results: Of the 62 RABs we analyzed in CRC, seven (RAB10, RAB11A, RAB15, RAB17, RAB19, RAB20, and RAB25) were significantly upregulated, while six (RAB6B, RAB9B, RAB12, RAB23, RAB31, and RAB34) were significantly downregulated in tumor tissues as compared to normal. We found that the upregulated-RABs, which were highly expressed in metabolic activated CRC subtype (CMS3), are associated with cell cycle related pathways enrichment and positively correlated with the mismatch repair (MMR) genes in CRC, implying their role in regulating cell metabolism and tumor growth. While, high expression of the downregulated-RABs were significantly associated with poor prognostic CRC mesenchymal subtypes (CMS4), immune checkpoint genes, and tumor infiltrating immune cells, indicating their role in predicting prognosis and immunotherapy efficacy. Interestingly, though RAB34 mRNA is downregulated in CRC, its high expression is significantly associated with poor prognosis. In vitro experiments showed that RAB17 overexpression can promote cell proliferation via cell cycle regulation. While, RAB34 overexpression can promote cell migration and invasion and is associated with PD-L1/PD-L2 expression increase in CRC cells. Conclusions: Our study showed that RABs may play important roles in regulating cell cycle and immune-related pathways, therefore might be potential biomarkers in predicting prognosis and immunotherapy response in CRC.

TSG-6 promotes Cancer Cell aggressiveness in a CD44-Dependent Manner and Reprograms Normal Fibroblasts to create a Pro-metastatic Microenvironment in Colorectal Cancer.

Tumor necrosis factor α stimulated gene 6 (TSG-6), a 30-KD secretory protein, plays an essential role in modulating inflammatory responses and extracellular matrix remodeling. However, little is known regarding the role of TSG-6 in human cancers. Here, we investigated the mechanism of action and the role of TSG-6 in colorectal cancer (CRC) metastasis. We found that TSG-6 was highly expressed in tumor tissues and was associated with poor prognosis and metastasis in CRC. Mechanistically, TSG-6 overexpression in CRC cells resulted in ERK activation and epithelial-mesenchymal transition by means of stabilizing CD44 and facilitating the CD44-EGFR complex formation on the cell membrane. Consequently, this resulted in the promotion of tumor migration and invasion both in vitro and in vivo. Notably, our data showed that CRC cells secreted TSG-6 could trigger a paracrine activation of JAK2-STAT3 signaling and reprogram normal fibroblasts into cancer-associated fibroblasts, which exhibited upregulation of pro-metastatic cytokines (CCL5 and MMP3) and higher movement ability. In animal models, the co-injection of cancer cells and TSG6-reprogrammed fibroblasts led to a significant increase in tumor metastasis. Our findings indicated that TSG-6 overexpression in CRC cells could promote cancer metastasis in both an autocrine and paracrine manner. Therefore, targeting TSG-6 might be a potential therapeutic strategy for the treatment of metastatic CRC.

NGFR Increases the Chemosensitivity of Colorectal Cancer Cells by Enhancing the Apoptotic and Autophagic Effects of 5-fluorouracil via the Activation of S100A9.

Colorectal cancer (CRC) is currently the third leading cause of cancer-related deaths worldwide, and 5-fluorouracil (5-FU)-based chemotherapies serve as important adjuvant therapies before and after surgery for CRC. However, the efficacy of CRC chemotherapy is limited by chemoresistance, and therefore the discovery of novel markers to indicate chemosensitivity is essential. Nerve growth factor receptor (NGFR), a cell surface receptor, is involved in cell death and survival. Our previous study indicated that NGFR acts as a tumor suppressor, and high expression is associated with better outcomes in patients receiving 5-FU-based adjuvant chemotherapy after surgery. The aim of this study was to investigate the effect of NGFR on the chemotherapeutic response in CRC. Chemosensitivity was investigated using DLD1 and HCT8 cells after NGFR transfection. Apoptosis was investigated by flow cytometry. Autophagy was assessed using GFP-LC3B transient transfection. Gene expression was measured using an mRNA microarray. Beclin-1 and Bcl-2 protein expressions were assessed by western blot. NGFR and S100 calcium-binding protein A9 (S100A9) expressions in CRC patients were investigated by immunohistochemistry. The results showed that the half maximal inhibitory concentration of NGFR-transfected cells was lower than that of controls in DLD1 and HCT8 cells after 5-FU treatment, and cell viability was lower than in empty-vector cells. Tumor sizes were also smaller than in empty-vector cells in vivo. The percentages of apoptotic and autophagic cells were higher in NGFR-transfected cells. NGFR elevated the expression of S100A9 after 5-FU treatment. The combination of Bcl-2 and Beclin-1 was significantly suppressed by overexpressed NGFR. Five-year overall and disease-free survival in NGFR+/S100A9+ patients was better than in NGFR-/S100A9- patients. This study's findings suggest that NGFR may serve as a marker predicting CRC patients' chemosensitivity.

Targeting and imaging colorectal cancer by activatable cell-penetrating peptides.

While it has been a great challenge to determine the positive status of metastasis lesions, intraoperative tumor imaging, which can show tumor localization and facilitate intraoperative staging of nodal metastases, have enabled surgeons to quickly and accurately perform radical resections. However, to date, there is no accurate method for evaluating nodal status intraoperatively. In this study, we synthesized activatable cell-penetrating peptides (ACPPs) that can specifically recognize colorectal cancer and their nodal status. ACPPs were labeled with Cy5 dye at the C-terminal, and named ACPP-Cy5. Laser scanning confocal microscopy and flow cytometry were used to measure the change in intracellular fluorescence intensity between cancer cells and normal cells. The results showed while the intracellular Cy5 fluorescent intensity can be visualized in both cancer and normal cells by 8 h after adding ACPP-Cy5, the relative fluorescence intensity of colorectal cancer cells was significantly higher than the normal cells. In addition, IVIS spectrum in vivo imaging system was used to observe the fluorescence intensity of ACPP-Cy5 after tail vein injection of mice with subcutaneous tumor or orthotopic colorectal cancer and liver metastasis. We found in mice with colorectal cancer and liver metastasis the Cy5 fluorescence intensity of cancer was significantly increased compared to the organs including liver, colorectum, lung, spleen, and heart. It is demonstrated here, this ACPPs can target colorectal cancer and liver metastasis, therefore ACPP-Cy5 may be a promising tool used for the diagnoses of colorectal cancer and to assist in tumor localization during surgery.

AQP9-induced cell cycle arrest is associated with RAS activation and improves chemotherapy treatment efficacy in colorectal cancer.

Aquaporin-9 (AQP9) expression is associated with arsenic sensitivity in leukemia cells. However, the role of AQP9 in regulating tumor sensitivity to adjuvant chemotherapy in colorectal cancer (CRC) has not been elucidated. In this study, we demonstrated that AQP9 can serve as an independent predictive marker for adjuvant chemotherapy in CRC. Patients with high AQP9 expression had higher rate of disease-free survival (DFS) than those with low AQP9 expression. Upregulation of AQP9 was associated with enhanced chemosensitivity to 5-fluorouracil (5-FU) both in vitro and in vivo. Overexpression of AQP9 resulted in an increased intracellular level of 5-FU in CRC cells, hence leading to a higher percentage of apoptosis after 5-FU treatment. Moreover, AQP9 is positively associated with RAS activation and other downstream signaling molecules in CRC. AQP9 overexpression resulted in p21 upregulation and induced S-phase arrest. Taken together, AQP9 enhances the cytotoxic response to 5-FU in CRC cells by simultaneously inducing S-phase arrest via activation of RAS signaling and facilitating drug uptake. Our results suggest that AQP9 might be a novel predictor for the benefit of 5-FU-based chemotherapy in CRC. The identification of AQP9-induced tumor sensitivity to 5-FU highlights the role of AQP9 in regulating chemosensitivity in CRC.