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.

Tumor derived exosomal ENTPD2 impair CD8+ T cell function in colon cancer through ATP-adenosine metabolism reprogramming.

BACKGROUND: Extracellular ATP-AMP-adenosine metabolism plays a pivotal role in modulating tumor immune responses. Previous studies have shown that the conversion of ATP to AMP is primarily catalysed by Ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1/CD39), a widely studied ATPase, which is expressed in tumor-associated immune cells. However, the function of ATPases derived from tumor cells themselves remains poorly understood. The purpose of this study was to investigate the role of colon cancer cell-derived ATPases in the development and progression of colon cancer. METHODS: Bioinformatic and tissue microarray analyses were performed to investigate the expression of ATPase family members in colon cancer. An ATP hydrolysis assay, high-performance liquid chromatography (HPLC), and CCK8 and colony formation assays were used to determine the effects of ENTPD2 on the biological functions of colon cancer cells. Flow cytometric and RNA-seq analyses were used to explore the function of CD8+ T cells. Immunoelectron microscopy and western blotting were used to evaluate the expression of ENTPD2 in exosomes. Double-labelling immunofluorescence and western blotting were used to examine the expression of ENTPD2 in serum exosomes and colon cancer tissues. RESULTS: We found that ENTPD2, rather than the well-known ATPase CD39, is highly expressed in cancer cells and is significantly positively associated with poor patient prognosis in patients with colon cancer. The overexpression of ENTPD2 in cancer cells augmented tumor progression in immunocompetent mice by inhibiting the function of CD8+ T cells. Moreover, ENTPD2 is localized primarily within exosomes. On the one hand, exosomal ENTPD2 reduces extracellular ATP levels, thereby inhibiting P2X7R-mediated NFATc1 nuclear transcription; on the other hand, it facilitates the increased conversion of ATP to adenosine, hence promoting adenosine-A2AR pathway activity. In patients with colon cancer, the serum level of exosomal ENTPD2 is positively associated with advanced TNM stage and high tumor invasion depth. Moreover, the level of ENTPD2 in the serum exosomes of colon cancer patients is positively correlated with the ENTPD2 expression level in paired colon cancer tissues, and the ENTPD2 level in both serum exosomes and tissues is significantly negatively correlated with the ENTPD2 expression level in tumor-infiltrating CD8+ T cells. CONCLUSION: Our study suggests that exosomal ENTPD2, originated from colon cancer cells, contributes to the immunosuppressive microenvironment by promoting ATP-adenosine metabolism. These findings highlight the importance of exosome-derived hydrolytic enzymes as independent entities in shaping the tumor immune microenvironment.

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.

HTRA1 from OVX rat osteoclasts causes detrimental effects on endplate chondrocytes through NF-κB.

Endplate osteochondritis is considered one of the major causes of intervertebral disc degeneration (IVDD) and low back pain. Menopausal women have a higher rate of endplate cartilage degeneration than similarly aged men, but the related mechanisms are still unclear. Subchondral bone changes, mainly mediated by osteoblasts and osteoclasts, are considered an important reason for the degeneration of cartilage. This work explored the role of osteoclasts in endplate cartilage degeneration, as well as its underlying mechanisms. A rat ovariectomy (OVX) model was used to induce estrogen deficiency. Our experiments indicated that OVX significantly promoted osteoclastogenesis and anabolism and catabolism changes in endplate chondrocytes. OVX osteoclasts cause an imbalance between anabolism and catabolism in endplate chondrocytes, as shown by a decrease in anabolic markers such as Aggrecan and Collagen II, and an increase in catabolic markers such as a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and matrix metalloproteinases (MMP13). Osteoclasts were also confirmed in this study to be able to secrete HtrA serine peptidase 1 (HTRA1), which resulted in increased catabolism in endplate chondrocytes through the NF-κB pathway under estrogen deficiency. This study demonstrated the involvement and mechanism of osteoclasts in the anabolism and catabolism changes of endplate cartilage under estrogen deficiency, and proposed a new strategy for the treatment of endplate osteochondritis and IVDD by targeting HTRA1.

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.

High-throughput proteomics profiling-derived signature associated with chemotherapy response and survival for stage II/III colorectal cancer.

Adjuvant chemotherapy (ACT) is usually used to reduce the risk of disease relapse and improve survival for stage II/III colorectal cancer (CRC). However, only a subset of patients could benefit from ACT. Thus, there is an urgent need to identify improved biomarkers to predict survival and stratify patients to refine the selection of ACT. We used high-throughput proteomics to analyze tumor and adjacent normal tissues of stage II/III CRC patients with /without relapse to identify potential markers for predicting prognosis and benefit from ACT. The machine learning approach was applied to identify relapse-specific markers. Then the artificial intelligence (AI)-assisted multiplex IHC was performed to validate the prognostic value of the relapse-specific markers and construct a proteomic-derived classifier for stage II/III CRC using 3 markers, including FHL3, GGA1, TGFBI. The proteomics profiling-derived signature for stage II/III CRC (PS) not only shows good accuracy to classify patients into high and low risk of relapse and mortality in all three cohorts, but also works independently of clinicopathologic features. ACT was associated with improved disease-free survival (DFS) and overall survival (OS) in stage II (pN0) patients with high PS and pN2 patients with high PS. This study demonstrated the clinical significance of proteomic features, which serve as a valuable source for potential biomarkers. The PS classifier provides prognostic value for identifying patients at high risk of relapse and mortality and optimizes individualized treatment strategy by detecting patients who may benefit from ACT for survival.

Associations of Homocysteine, Folate, and Vitamin B12 with Osteoarthritis: A Mendelian Randomization Study.

Homocysteine, inversely related to folate and vitamin B12, is an independent risk factor for several age-related disorders. However, little is known about the association of homocysteine and related vitamins with osteoarthritis (OA). This study aimed to elucidate the potential causal effects of homocysteine, folate, and vitamin B12 on site- and gender-specific OA by applying the two-sample Mendelian randomization (MR) approach. Genetically predicted homocysteine showed adverse effects on overall OA (95% confidence interval (CI): 1.044-1.155), knee OA (95% CI: 1.000-1.167), hip OA (95% CI: 1.057-1.297), and spine OA (95% CI: 1.017-1.216). Genetically predicted folate showed protective effects on overall OA (95% CI: 0.783-0.961) and spine OA (95% CI: 0.609-0.954). Folate (95% CI: 0.887-1.004) and vitamin B12 (95% CI: 0.886-1.009) showed a protective trend against knee OA. The patterns of associations were site and gender specific. In conclusion, homocysteine had adverse effects on OA, especially on OA at weight-bearing joints and in females. Folate and vitamin B12 had protective effects on OA. Homocysteine-lowering interventions may be a potential option in the treatment and prevention of OA.

CTGF as a multifunctional molecule for cartilage and a potential drug for osteoarthritis.

CTGF is a multifunctional protein and plays different roles in different cells and under different conditions. Pamrevlumab, a monoclonal antibody against CTGF, is an FDA approved drug for idiopathic pulmonary fibrosis (IPF) and Duchenne muscular dystrophy (DMD). Recent studies have shown that CTGF antibodies may potentially serve as a new drug for osteoarthritis (OA). Expression of CTGF is significantly higher in OA joints than in healthy counterparts. Increasing attention has been attracted due to its interesting roles in joint homeostasis. Joint homeostasis relies on normal cellular functions and cell-cell interactions. CTGF is essential for physiological activities of chondrocytes. Abnormal CTGF expression may cause cartilage degeneration. In this review, the physiological functions of CTGF in chondrocytes and related mechanisms are summarized. Changes in the related signaling pathways due to abnormal CTGF are discussed, which are contributing factors to inflammation, cartilage degeneration and synovial fibrosis in OA. The possibility of CTGF as a potential therapeutic target for OA treatment are reviewed.

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.

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.

Distinct pathological changes of osteochondral units in early OVX-OA involving TGF-ß signaling.

Introduction: Different opinions exist about the role of subchondral bone in osteoarthritis (OA), probably because subchondral bone has different effects on cartilage degeneration in OA induced by different pathologies. Animal studies to illustrate the role of subchondral bone in cartilage degeneration were mostly based on post-traumatic OA (PT-OA). Postmenopausal women experience a much higher occurrence of OA than similar-aged men. The physiological changes and pathogenesis of the osteochondral unit in ovariectomy-induced OA (OVX-OA) might be distinct from other types of OA. Methods: The osteochondral alterations of post-traumatic OA (PT-OA) and OVX-OA at week 9 after surgery were compared. Then the alterations of osteochondral units in OVX-OA rats were tracked over time for the designed groups: Sham, OVX and OVX rats treated with estrogen (OVX+E). DXA, micro-CT, and histochemical staining were performed to observe alterations in osteochondral units. Results: Rapid cartilage degeneration and increased bone formation were observed in PT-OA, while only mild cartilage erosion and significant bone loss were observed in OVX-OA at week 9 after surgery. Subchondral bone degradation preceded cartilage degeneration by 6 weeks in OVX-OA. TGF-ß expression was downregulated in the osteochondral unit of OVX rats. Estrogen supplementation inhibited subchondral bone loss, cartilage degradation and TGF-ß expression decrease. Discussion: This research demonstrated the distinct behaviors of the osteochondral unit and the critical role of subchondral bone in early OVX-OA compared with PT-OA. Inhibiting subchondral bone catabolism at the early stage of OVX-OA could be an effective treatment for post-menopausal OA. Based on the results, estrogen supplementation and TGF-ß modulation at the early stage are both potential therapies for post-menopausal OA.

Inhibition of the PLK1-Coupled Cell Cycle Machinery Overcomes Resistance to Oxaliplatin in Colorectal Cancer.

Dysregulation of the cell cycle machinery leads to genomic instability and is a hallmark of cancer associated with chemoresistance and poor prognosis in colorectal cancer (CRC). Identifying and targeting aberrant cell cycle machinery is expected to improve current therapies for CRC patients. Here,upregulated polo-like kinase 1 (PLK1) signaling, accompanied by deregulation of cell cycle-related pathways in CRC is identified. It is shown that aberrant PLK1 signaling correlates with recurrence and poor prognosis in CRC patients. Genetic and pharmacological blockade of PLK1 significantly increases the sensitivity to oxaliplatin in vitro and in vivo. Mechanistically, transcriptomic profiling analysis reveals that cell cycle-related pathways are activated by oxaliplatin treatment but suppressed by a PLK1 inhibitor. Cell division cycle 7 (CDC7) is further identified as a critical downstream effector of PLK1 signaling, which is transactivated via the PLK1-MYC axis. Increased CDC7 expression is also found to be positively correlated with aberrant PLK1 signaling in CRC and is associated with poor prognosis. Moreover, a CDC7 inhibitor synergistically enhances the anti-tumor effect of oxaliplatin in CRC models, demonstrating the potential utility of targeting the PLK1-MYC-CDC7 axis in the treatment of oxaliplatin-based chemotherapy.

Stromal induction of BRD4 phosphorylation Results in Chromatin Remodeling and BET inhibitor Resistance in Colorectal Cancer.

BRD4, a Bromodomain and Extraterminal (BET) protein family member, is a promising anti-cancer drug target. However, resistance to BET inhibitors targeting BRD4 is common in solid tumors. Here, we show that cancer-associated fibroblast (CAF)-activated stromal signaling, interleukin-6/8-JAK2, induces BRD4 phosphorylation at tyrosine 97/98 in colorectal cancer, resulting in BRD4 stabilization due to interaction with the deubiquitinase UCHL3. BRD4 phosphorylation at tyrosine 97/98 also displays increased binding to chromatin but reduced binding to BET inhibitors, resulting in resistance to BET inhibitors. We further show that BRD4 phosphorylation promotes interaction with STAT3 to induce chromatin remodeling through concurrent binding to enhancers and super-enhancers, supporting a tumor-promoting transcriptional program. Inhibition of IL6/IL8-JAK2 signaling abolishes BRD4 phosphorylation and sensitizes BET inhibitors in vitro and in vivo. Our study reveals a stromal mechanism for BRD4 activation and BET inhibitor resistance, which provides a rationale for developing strategies to treat CRC more effectively.

Alterations in bile acid metabolizing gut microbiota and specific bile acid genes as a precision medicine to subclassify NAFLD.

Multiple mechanisms for the gut microbiome contributing to the pathogenesis of nonalcoholic fatty liver disease (NAFLD) have been implicated. Here, we aim to investigate the contribution and potential application for altered bile acids (BA) metabolizing microbes in NAFLD by post hoc analysis of whole metagenome sequencing (WMS) data. The discovery cohort consisted of 86 well-characterized patients with biopsy-proven NAFLD and 38 healthy controls. Assembly-based analysis was performed to identify BA-metabolizing microbes. Statistical tests, feature selection, and microbial coabundance analysis were integrated to identify microbial alterations and markers in NAFLD. An independent validation cohort was subjected to similar analyses. NAFLD microbiota exhibited decreased diversity and microbial associations. We established a classifier model with 53 differential species exhibiting a robust diagnostic accuracy [area under the receiver-operator curve (AUC) = 0.97] for detecting NAFLD. Next, eight important differential pathway markers including secondary BA biosynthesis were identified. Specifically, increased abundance of 7α-hydroxysteroid dehydrogenase (7α-HSDH), 3α-hydroxysteroid dehydrogenase (baiA), and bile acid-coenzyme A ligase (baiB) was detected in NAFLD. Furthermore, 10 of 50 BA-metabolizing metagenome-assembled genomes (MAGs) from Bacteroides ovatus and Eubacterium biforme were dominant in NAFLD and interplayed as a synergetic ecological guild. Importantly, two subtypes of patients with NAFLD were observed according to secondary BA metabolism potentials. Elevated capability for secondary BA biosynthesis was also observed in the validation cohort. These bacterial BA-metabolizing genes and microbes identified in this study may serve as disease markers. Microbial differences in BA-metabolism and strain-specific differences among patients highlight the potential for precision medicine in NAFLD treatment.

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.

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.

Ubiquitin ligase TRIM65 promotes colorectal cancer metastasis by targeting ARHGAP35 for protein degradation.

Tripartite motif-containing protein 65 (TRIM65) is an E3 ubiquitin ligase and a critical regulator of a variety of cellular processes as well as tumor progression. Therefore, more substrates must be identified in the physiology or disease context. Here, we found that TRIM65 is upregulated and associated with poor survival in colorectal cancer (CRC). More specifically, high expression of TRIM65 is associated with CRC metastasis and recurrence. Ectopic overexpression of TRIM65 in CRC cell lines enhanced proliferation, invasion, and migration, while knockdown of TRIM65 expression had the opposite effects. Furthermore, we identified a new substrate of TRIM65, namely ARHGAP35, a Rho GTPase-activating protein (GAP) that is involved in polarized cell migration. Phenotypically, forced expression of TRIM65 induces increased production of migration-related structures, focal adhesions, and/or filopodia and enhances CRC metastasis to the liver or the lung in a mouse model. Mechanistic studies revealed that TRIM65 mediates ubiquitination of ARHGAP35, whose degradation leads to elevated Rho GTPase activity. In addition, we identified several phosphorylation sites on TRIM65. In sum, we reveal a novel TRIM65-GAP-Rho regulatory axis that modulates the actin cytoskeleton and the migration behavior of CRC cells, and the TRIM65-ARHGAP35 interaction might be a valuable therapeutic target in CRC.

The novel long noncoding RNA u50535 promotes colorectal cancer growth and metastasis by regulating CCL20.

Long noncoding RNAs (lncRNAs) have been emerging as master regulators of tumor growth and metastasis, but the functions and underlying mechanisms of lncRNAs in colorectal cancer (CRC) still need to be clarified. Here, we found a novel lncRNA u50535, which was greatly overexpressed in CRC tissues and was associated with poor prognosis in CRC patients. Function studies showed that u50535 was an oncogene in CRC both in vitro and in vivo. In mechanism, through RNA sequencing and rescue assay, we found that u50535 activates CCL20 signaling to promote cell proliferation and migration in CRC. Taken together, these findings suggest that u50535 can promote CRC growth and metastasis and may serve as a potential biomarker in CRC.

Hypoxic tumor microenvironment activates GLI2 via HIF-1α and TGF-ß2 to promote chemoresistance in colorectal cancer.

Colorectal cancer patients often relapse after chemotherapy, owing to the survival of stem or progenitor cells referred to as cancer stem cells (CSCs). Although tumor stromal factors are known to contribute to chemoresistance, it remains not fully understood how CSCs in the hypoxic tumor microenvironment escape the chemotherapy. Here, we report that hypoxia-inducible factor (HIF-1α) and cancer-associated fibroblasts (CAFs)-secreted TGF-ß2 converge to activate the expression of hedgehog transcription factor GLI2 in CSCs, resulting in increased stemness/dedifferentiation and intrinsic resistance to chemotherapy. Genetic or small-molecule inhibitor-based ablation of HIF-1α/TGF-ß2-mediated GLI2 signaling effectively reversed the chemoresistance caused by the tumor microenvironment. Importantly, high expression levels of HIF-1α/TGF-ß2/GLI2 correlated robustly with the patient relapse following chemotherapy, highlighting a potential biomarker and therapeutic target for chemoresistance in colorectal cancer. Our study thus uncovers a molecular mechanism by which hypoxic colorectal tumor microenvironment promotes cancer cell stemness and resistance to chemotherapy and suggests a potentially targeted treatment approach to mitigating chemoresistance.

Downregulation of TRIM58 expression is associated with a poor patient outcome and enhances colorectal cancer cell invasion.

TRIM58 is a member of the tripartite motif protein (TRIM) family of E3 ubiquitin ligases. Aberrant gene methylation of TRIM58 has been reported in liver and lung cancer and indicates a poor patient prognosis. However, the expression level and functional role of TRIM58 in colorectal cancer (CRC) have yet to be elucidated. In the present study, we found that TRIM58 expression was significantly suppressed in human CRC and was inversely correlated with CRC progression. Additionally, overall survival was significantly reduced in patients with low TRIM58 expression in CRC tumors. In vitro studies demonstrated that ectopic TRIM58 overexpression strongly inhibited CRC cell invasion but had minimal effects on cell proliferation, colonization and migration. Furthermore, TRIM58 suppression enhanced the expression of epithelial-to-mesenchymal transition (EMT) and matrix metalloproteinase (MMP) genes. Thus, our findings suggest that TRIM58 is a potential prognostic marker of CRC and functions as a tumor-suppressor gene via inhibition of cancer cell invasion through EMT and MMP activation.