Albendazole inhibits colon cancer progression and therapy resistance by targeting ubiquitin ligase RNF20.

BACKGROUND: The repurposing of FDA-approved drugs for anti-cancer therapies is appealing due to their established safety profiles and pharmacokinetic properties and can be quickly moved into clinical trials. Cancer progression and resistance to conventional chemotherapy remain the key hurdles in improving the clinical management of colon cancer patients and associated mortality. METHODS: High-throughput screening (HTS) was performed using an annotated library of 1,600 FDA-approved drugs to identify drugs with strong anti-CRC properties. The candidate drug exhibiting most promising inhibitory effects in in-vitro studies was tested for its efficacy using in-vivo models of CRC progression and chemoresistance and patient derived organoids (PTDOs). RESULTS: Albendazole, an anti-helminth drug, demonstrated the strongest inhibitory effects on the tumorigenic potentials of CRC cells, xenograft tumor growth and organoids from mice. Also, albendazole sensitized the chemoresistant CRC cells to 5-fluorouracil (5-FU) and oxaliplatin suggesting potential to treat chemoresistant CRC. Mechanistically, Albendazole treatment modulated the expression of RNF20, to promote apoptosis in CRC cells by delaying the G2/M phase and suppressing anti-apoptotic-Bcl2 family transcription. CONCLUSIONS: Albendazole, an FDA approved drug, carries strong therapeutic potential to treat colon cancers which are aggressive and potentially resistant to conventional chemotherapeutic agents. Our findings also lay the groundwork for further clinical testing.

Claudin-1 interacts with EPHA2 to promote cancer stemness and chemoresistance in colorectal cancer.

Therapy resistance is the primary problem in treating late-stage colorectal cancer (CRC). Claudins are frequently dysregulated in cancer, and several are being investigated as novel therapeutic targets and biomarkers. We have previously demonstrated that Claudin-1 (CLDN1) expression in CRC promotes epithelial-mesenchymal transition, metastasis, and resistance to anoikis. Here, we hypothesize that CLDN1 promotes cancer stemness and chemoresistance in CRC. We found that high CLDN1 expression in CRC is associated with cancer stemness and chemoresistance signaling pathways in patient datasets, and it promotes chemoresistance both in vitro and in vivo. Using functional stemness assays, proteomics, biophysical binding assays, and patient-derived organoids, we found that CLDN1 promotes properties of cancer stemness including CD44 expression, tumor-initiating potential, and chemoresistance through a direct interaction with ephrin type-A receptor 2 (EPHA2) tyrosine kinase. This interaction is dependent on the CLDN1 PDZ-binding motif, increases EPHA2 protein expression by inhibiting its degradation, and enhances downstream AKT signaling and CD44 expression to promote stemness and chemoresistance. These results suggest CLDN1 is a viable target for pharmacological intervention and/or biomarker development.

Identification and characterization of a first-generation inhibitor of claudin-1 in colon cancer progression and metastasis.

Colorectal cancer (CRC) is a leading cause of the cancer-related deaths worldwide. Thus, developing novel and targeted therapies for inhibiting CRC progression and metastasis is urgent. Several studies, including ours, have reported a causal role for an upregulated claudin-1 expression in promoting CRC metastasis through the activation of the Src and ß-catenin-signaling. In murine models of colon tumorigenesis, claudin-1 overexpression promotes oncogenic properties such as transformation and invasiveness. Conversely, the downregulation of claudin-1 inhibits colon tumorigenesis. Despite being a desirable target for cancer treatment, there are currently no known claudin-1 inhibitors with antitumor efficacy. Using a rigorous analytical design and implementing in- vitro and in-vivo testing and a brief medicinal chemistry campaign, we identified a claudin-1-specific inhibitor and named it I-6. Despite its high potency, I-6 was rapidly cleared in human liver microsomes. We, therefore, synthesized I-6 analogs and discovered a novel small molecule, PDS-0330. We determined that PDS0330 inhibits claudin-1-dependent CRC progression without exhibiting toxicity in in-vitro and in-vivo models of CRC and that it binds directly and specifically to claudin-1 with micromolar affinity. Further analyses revealed that PDS-0330 exhibits antitumor and chemosensitizer activities with favorable pharmacokinetic properties by inhibiting the association with metastatic oncogene Src. Overall, our data propose that PDS-0330 interferes with claudin-1/Src association to inhibit CRC progression and metastasis. Our findings are of direct clinical relevance and may open new therapeutic opportunities in colon cancer treatment and/or management by targeting claudin-1.

Role of CD44 isoforms in epithelial-mesenchymal plasticity and metastasis.

Cellular plasticity lies at the core of cancer progression, metastasis, and resistance to treatment. Stemness and epithelial-mesenchymal plasticity in cancer are concepts that represent a cancer cell's ability to coopt and adapt normal developmental programs to promote survival and expansion. The cancer stem cell model states that a small subset of cancer cells with stem cell-like properties are responsible for driving tumorigenesis and metastasis while remaining especially resistant to common chemotherapeutic drugs. Epithelial-mesenchymal plasticity describes a cancer cell's ability to transition between epithelial and mesenchymal phenotypes which drives invasion and metastasis. Recent research supports the existence of stable epithelial/mesenchymal hybrid phenotypes which represent highly plastic states with cancer stem cell characteristics. The cell adhesion molecule CD44 is a widely accepted marker for cancer stem cells, and it lies at a functional intersection between signaling networks regulating both stemness and epithelial-mesenchymal plasticity. CD44 expression is complex, with alternative splicing producing many isoforms. Interestingly, not only does the pattern of isoform expression change during transitions between epithelial and mesenchymal phenotypes in cancer, but these isoforms have distinct effects on cell behavior including the promotion of metastasis and stemness. The role of CD44 both downstream and upstream of signaling pathways regulating epithelial-mesenchymal plasticity and stemness make this protein a valuable target for further research and therapeutic intervention.

A Claudin-Based Molecular Signature Identifies High-Risk, Chemoresistant Colorectal Cancer Patients.

Identifying molecular characteristics that are associated with aggressive cancer phenotypes through gene expression profiling can help predict treatment responses and clinical outcomes. Claudins are deregulated in colorectal cancer (CRC). In CRC, increased claudin-1 expression results in epithelial-to-mesenchymal transition and metastasis, while claudin-7 functions as a tumor suppressor. In this study, we have developed a molecular signature based on claudin-1 and claudin-7 associated with poor patient survival and chemoresistance. This signature was validated using an integrated approach including publicly available datasets and CRC samples from patients who either responded or did not respond to standard-of-care treatment, CRC cell lines, and patient-derived rectal and colon tumoroids. Transcriptomic analysis from a patient dataset initially yielded 23 genes that were differentially expressed along with higher claudin-1 and decreased claudin-7. From this analysis, we selected a claudins-associated molecular signature including PIK3CA, SLC6A6, TMEM43, and ASAP-1 based on their importance in CRC. The upregulation of these genes and their protein products was validated using multiple CRC patient datasets, in vitro chemoresistant cell lines, and patient-derived tumoroid models. Additionally, blocking these genes improved 5-FU sensitivity in chemoresistant CRC cells. Our findings propose a new claudin-based molecular signature that associates with poor prognosis as well as characteristics of treatment-resistant CRC including chemoresistance, metastasis, and relapse.

PIK3C3 Inhibition Promotes Sensitivity to Colon Cancer Therapy by Inhibiting Cancer Stem Cells.

Background: Despite recent advances in therapies, resistance to chemotherapy remains a critical problem in the clinical management of colorectal cancer (CRC). Cancer stem cells (CSCs) play a central role in therapy resistance. Thus, elimination of CSCs is crucial for effective CRC therapy; however, such strategies are limited. Autophagy promotes resistance to cancer therapy; however, whether autophagy protects CSCs to promote resistance to CRC-therapy is not well understood. Moreover, specific and potent autophagy inhibitors are warranted as clinical trials with hydroxychloroquine have not been successful. Methods: Colon cancer cells and tumoroids were used. Fluorescent reporter-based analysis of autophagy flux, spheroid and side population (SP) culture, and qPCR were done. We synthesized 36-077, a potent inhibitor of PIK3C3/VPS34 kinase, to inhibit autophagy. Combination treatments were done using 5-fluorouracil (5-FU) and 36-077. Results: The 5-FU treatment induced autophagy only in a subset of the treated colon cancer. These autophagy-enriched cells also showed increased expression of CSC markers. Co-treatment with 36-077 significantly improved efficacy of the 5-FU treatment. Mechanistic studies revealed that combination therapy inhibited GSK-3ß/Wnt/ß-catenin signaling to inhibit CSC population. Conclusion: Autophagy promotes resistance to CRC-therapy by specifically promoting GSK-3ß/Wnt/ß-catenin signaling to promote CSC survival, and 36-077, a PIK3C3/VPS34 inhibitor, helps promote efficacy of CRC therapy.

Role of Claudin Proteins in Regulating Cancer Stem Cells and Chemoresistance-Potential Implication in Disease Prognosis and Therapy.

Claudins are cell-cell adhesion proteins, which are expressed in tight junctions (TJs), the most common apical cell-cell adhesion. Claudin proteins help to regulate defense and barrier functions, as well as differentiation and polarity in epithelial and endothelial cells. A series of studies have now reported dysregulation of claudin proteins in cancers. However, the precise mechanisms are still not well understood. Nonetheless, studies have clearly demonstrated a causal role of multiple claudins in the regulation of epithelial to mesenchymal transition (EMT), a key feature in the acquisition of a cancer stem cell phenotype in cancer cells. In addition, claudin proteins are known to modulate therapy resistance in cancer cells, a feature associated with cancer stem cells. In this review, we have focused primarily on highlighting the causal link between claudins, cancer stem cells, and therapy resistance. We have also contemplated the significance of claudins as novel targets in improving the efficacy of cancer therapy. Overall, this review provides a much-needed understanding of the emerging role of claudin proteins in cancer malignancy and therapeutic management.

Correction: Upregulated claudin-1 expression promotes colitis-associated cancer by promoting ß-catenin phosphorylation and activation in Notch/p-AKT-dependent manner.

In Fig. 1 legend, should read as follows: Cld-1 intensifies colitis, leads to impaired recovery from DSS induction and triggers dysplasia: a Schematic illustration of the experimental protocol. Age-matched and sex-matched WT (n = 8) and Cld-1 Tg (n = 8) mice were treated with 2.5% DSS in drinking water for 7 days ad libitum (colitis group), followed by drinking water for 10 days (DSS recovery group). b Representative histological images of WT and Cld-1 Tg mice under DSS and DSS recovery protocol showing regenerative crypts in WT DSS Recovery and dysplastic crypts in Cld-1 Tg Recovery. c The mean changes in body weight of the WT and Cld-1 Tg mice after being fed with 2.5% DSS were measured every day until day 7 for the colitis group and day 10 for the DSS recovery group. The percentage of mice possessing dysplastic crypt in the recovery protocol among WT and Cld-1 Tg groups. Results are statistically significant at p < 0.05. Values sharing following symbols differ significantly, asterisk (*) compared with WT DSS Recovery.

Upregulated claudin-1 expression promotes colitis-associated cancer by promoting ß-catenin phosphorylation and activation in Notch/p-AKT-dependent manner.

In IBD patients, integration between a hyper-activated immune system and epithelial cell plasticity underlies colon cancer development. However, molecular regulation of such a circuity remains undefined. Claudin-1 (Cld-1), a tight-junction integral protein deregulation alters colonic epithelial cell (CEC) differentiation, and promotes colitis severity while impairing colitis-associated injury/repair. Tumorigenesis is a product of an unregulated wound-healing process and therefore we postulated that upregulated Cld-1 levels render IBD patients susceptible to the colitis-associated cancer (CAC). Villin Cld-1 mice are used to carryout overexpressed studies in mice. The role of deregulated Cld-1 expression in CAC and the underlying mechanism was determined using a well-constructed study scheme and mouse models of DSS colitis/recovery and CAC. Using an inclusive investigative scheme, we here report that upregulated Cld-1 expression promotes susceptibility to the CAC and its malignancy. Increased mucosal inflammation and defective epithelial homeostasis accompanied the increased CAC in Villin-Cld-1-Tg mice. We further found significantly increased levels of protumorigenic M2 macrophages and ß-cateninSer552 (ß-CatSer552) expression in the CAC in Cld-1Tg vs. WT mice. Mechanistic studies identified the role of PI3K/Akt signaling in Cld-1-dependent activation of the ß-CatSer552, which, in turn, was dependent on proinflammatory signals. Our studies identify a critical role of Cld-1 in promoting susceptibility to CAC. Importantly, these effects of deregulated Cld-1 were not associated with altered tight junction integrity, but on its noncanonical role in regulating Notch/PI3K/Wnt/ ß-CatSer552 signaling. Overall, outcome from our current studies identifies Cld-1 as potential prognostic biomarker for IBD severity and CAC, and a novel therapeutic target.

MASTL induces Colon Cancer progression and Chemoresistance by promoting Wnt/ß-catenin signaling.

BACKGROUND: Chemotherapeutic agents that modulate cell cycle checkpoints and/or tumor-specific pathways have shown immense promise in preclinical and clinical studies aimed at anti-cancer therapy. MASTL (Greatwall in Xenopus and Drosophila), a serine/threonine kinase controls the final G2/M checkpoint and prevents premature entry of cells into mitosis. Recent studies suggest that MASTL expression is highly upregulated in cancer and confers resistance against chemotherapy. However, the role and mechanism/s of MASTL mediated regulation of tumorigenesis remains poorly understood. METHODS: We utilized a large patient cohort and mouse models of colon cancer as well as colon cancer cells to determine the role of Mastl and associated mechanism in colon cancer. RESULTS: Here, we show that MASTL expression increases in colon cancer across all cancer stages compared with normal colon tissue (P < 0.001). Also, increased levels of MASTL associated with high-risk of the disease and poor prognosis. Further, the shRNA silencing of MASTL expression in colon cancer cells induced cell cycle arrest and apoptosis in vitro and inhibited xenograft-tumor growth in vivo. Mechanistic analysis revealed that MASTL expression facilitates colon cancer progression by promoting the ß-catenin/Wnt signaling, the key signaling pathway implicated in colon carcinogenesis, and up-regulating anti-apoptotic proteins, Bcl-xL and Survivin. Further studies where colorectal cancer (CRC) cells were subjected to 5-fluorouracil (5FU) treatment revealed a sharp increase in MASTL expression upon chemotherapy, along with increases in Bcl-xL and Survivin expression. Most notably, inhibition of MASTL in these cells induced chemosensitivity to 5FU with downregulation of Survivin and Bcl-xL expression. CONCLUSION: Overall, our data shed light on the heretofore-undescribed mechanistic role of MASTL in key oncogenic signaling pathway/s to regulate colon cancer progression and chemo-resistance that would tremendously help to overcome drug resistance in colon cancer treatment.