Treatment of HCC with claudin-1-specific antibodies suppresses carcinogenic signaling and reprograms the tumor microenvironment.

BACKGROUND & AIMS: Despite recent approvals, the response to treatment and prognosis of patients with advanced hepatocellular carcinoma (HCC) remain poor. Claudin-1 (CLDN1) is a membrane protein that is expressed at tight junctions, but it can also be exposed non-junctionally, such as on the basolateral membrane of the human hepatocyte. While CLDN1 within tight junctions is well characterized, the role of non-junctional CLDN1 and its role as a therapeutic target in HCC remains unexplored. METHODS: Using humanized monoclonal antibodies (mAbs) specifically targeting the extracellular loop of human non-junctional CLDN1 and a large series of patient-derived cell-based and animal model systems we aimed to investigate the role of CLDN1 as a therapeutic target for HCC. RESULTS: Targeting non-junctional CLDN1 markedly suppressed tumor growth and invasion in cell line-based models of HCC and patient-derived 3D ex vivo models. Moreover, the robust effect on tumor growth was confirmed in vivo in a large series of cell line-derived xenograft and patient-derived xenograft mouse models. Mechanistic studies, including single-cell RNA sequencing of multicellular patient HCC tumorspheres, suggested that CLDN1 regulates tumor stemness, metabolism, oncogenic signaling and perturbs the tumor immune microenvironment. CONCLUSIONS: Our results provide the rationale for targeting CLDN1 in HCC and pave the way for the clinical development of CLDN1-specific mAbs for the treatment of advanced HCC. IMPACT AND IMPLICATIONS: Hepatocellular carcinoma (HCC) is associated with high mortality and unsatisfactory treatment options. Herein, we identified the cell surface protein Claudin-1 as a treatment target for advanced HCC. Monoclonal antibodies targeting Claudin-1 inhibit tumor growth in patient-derived ex vivo and in vivo models by modulating signaling, cell stemness and the tumor immune microenvironment. Given the differentiated mechanism of action, the identification of Claudin-1 as a novel therapeutic target for HCC provides an opportunity to break the plateau of limited treatment response. The results of this preclinical study pave the way for the clinical development of Claudin-1-specific antibodies for the treatment of advanced HCC. It is therefore of key impact for physicians, scientists and drug developers in the field of liver cancer and gastrointestinal oncology.

Exploiting the predictive power of educated spheroids to detect immune-mediated idiosyncratic drug-induced liver injury: the case of troglitazone.

Idiosyncratic drug-induced liver injury (iDILI) is a major concern in drug development because its occurrence is unpredictable. Presently, iDILI prediction is a challenge, and cell toxicity is observed only at concentrations that are much higher than the therapeutic doses in preclinical models. Applying a proprietary cell educating technology, we developed a person-dependent spheroid system that contains autologous educated immune cells that can detect iDILI risk at therapeutic concentrations. Integrating this system into a high-throughput screening platform will help pharmaceutical companies accurately detect the iDILI risk of new molecules de-risking drug development.

Improving the response to oxaliplatin by targeting chemotherapy-induced CLDN1 in resistant metastatic colorectal cancer cells.

BACKGROUND: Tumor resistance is a frequent cause of therapy failure and remains a major challenge for the long-term management of colorectal cancer (CRC). The aim of this study was to determine the implication of the tight junctional protein claudin 1 (CLDN1) in the acquired resistance to chemotherapy. METHODS: Immunohistochemistry was used to determine CLDN1 expression in post-chemotherapy liver metastases from 58 CRC patients. The effects of oxaliplatin on membrane CLDN1 expression were evaluated by flow cytometry, immunofluorescence and western blotting experiments in vitro and in vivo. Phosphoproteome analyses, proximity ligation and luciferase reporter assays were used to unravel the mechanism of CLDN1 induction. RNAseq experiments were performed on oxaliplatin-resistant cell lines to investigate the role of CLDN1 in chemoresistance. The "one-two punch" sequential combination of oxaliplatin followed by an anti-CLDN1 antibody-drug conjugate (ADC) was tested in both CRC cell lines and murine models. RESULTS: We found a significant correlation between CLDN1 expression level and histologic response to chemotherapy, CLDN1 expression being the highest in resistant metastatic residual cells of patients showing minor responses. Moreover, in both murine xenograft model and CRC cell lines, CLDN1 expression was upregulated after exposure to conventional chemotherapies used in CRC treatment. CLDN1 overexpression was, at least in part, functionally related to the activation of the MAPKp38/GSK3ß/Wnt/ß-catenin pathway. Overexpression of CLDN1 was also observed in oxaliplatin-resistant CRC cell lines and was associated with resistance to apoptosis, suggesting an anti-apoptotic role for CLDN1. Finally, we demonstrated that the sequential treatment with oxaliplatin followed by an anti-CLDN1 ADC displayed a synergistic effect in vitro and in in vivo. CONCLUSION: Our study identifies CLDN1 as a new biomarker of acquired resistance to chemotherapy in CRC patients and suggests that a "one-two punch" approach targeting chemotherapy-induced CLDN1 expression may represent a therapeutic opportunity to circumvent resistance and to improve the outcome of patients with advanced CRC.

A monoclonal antibody targeting nonjunctional claudin-1 inhibits fibrosis in patient-derived models by modulating cell plasticity.

Tissue fibrosis is a key driver of end-stage organ failure and cancer, overall accounting for up to 45% of deaths in developed countries. There is a large unmet medical need for antifibrotic therapies. Claudin-1 (CLDN1) is a member of the tight junction protein family. Although the role of CLDN1 incorporated in tight junctions is well established, the function of nonjunctional CLDN1 (njCLDN1) is largely unknown. Using highly specific monoclonal antibodies targeting a conformation-dependent epitope of exposed njCLDN1, we show in patient-derived liver three-dimensional fibrosis and human liver chimeric mouse models that CLDN1 is a mediator and target for liver fibrosis. Targeting CLDN1 reverted inflammation-induced hepatocyte profibrogenic signaling and cell fate and suppressed the myofibroblast differentiation of hepatic stellate cells. Safety studies of a fully humanized antibody in nonhuman primates did not reveal any serious adverse events even at high steady-state concentrations. Our results provide preclinical proof of concept for CLDN1-specific monoclonal antibodies for the treatment of advanced liver fibrosis and cancer prevention. Antifibrotic effects in lung and kidney fibrosis models further indicate a role of CLDN1 as a therapeutic target for tissue fibrosis across organs. In conclusion, our data pave the way for further therapeutic exploration of CLDN1-targeting therapies for fibrotic diseases in patients.

Claudin gene expression profiles and clinical value in colorectal tumors classified according to their molecular subtype.

PURPOSE: Colorectal cancer (CRC) is a heterogeneous disease that can be classified into distinct molecular subtypes. The aims of this study were 1) to compare claudin (CLDN) gene expression in CRC samples and normal colon mucosa, and then in the different CRC molecular subtypes, and 2) to assess their prognostic value. PATIENTS AND METHODS: CLDN expression in CRC samples was analyzed using gene expression data for a cohort of 143 primary CRC samples, and compared in the same CRC samples classified into different molecular subtypes (C1 to C6 according to the Marisa's classification, and CMS1 to CMS4 of the consensus classification). Comparison of CLDN expression in normal and tumor colon samples was also made on a smaller number of samples. Then, the relationship between CLDN expression profiles and overall survival (OS) and progression-free survival was examined. RESULTS: Compared with normal mucosa, CLDN1 and CLDN2 were upregulated, whereas CLDN5, 7, 8, and 23 were downregulated in CRC samples. Variations in CLDN expression profiles were observed mainly in the CMS2/C1 and CMS4/C4 subtypes. Overall, expression of CLDN2 or CLDN4 alone had a strong prognostic value that increased when they were associated. In the CMS4/C4 subtypes, lower expressions of CLDN11, CLDN12, and CLDN23 were associated with longer OS. Conversely, in the CMS2 and C1 subtypes, low CLDN23 expression was associated with shorter OS and progression-free survival, suggesting a dual role for CLDN23 as a tumor suppressor/promoter in CRC. CLDN6 and CLDN11 had a prognostic value in the CMS2 and C4 subtypes, respectively. CONCLUSION: This analysis of CLDN gene expression profiles and prognostic value in CRC samples classified according to their molecular subtype shows that CRC heterogeneity must be taken into account when assessing CLDN potential value as prognostic markers or therapeutic targets.

PKCθ-induced phosphorylations control the ability of Fra-1 to stimulate gene expression and cancer cell migration.

The AP-1 transcription factor Fra-1 is aberrantly expressed in a large number of cancers and plays crucial roles in cancer development and progression by stimulating the expression of genes involved in these processes. However, the control of Fra-1 transactivation ability is still unclear and here we hypothesized that PKCθ-induced phosphorylation could be necessary to obtain a fully active Fra-1 protein. Using MCF7 stable cells overexpressing equivalent levels of unphosphorylated Fra-1 or PKCθ-phosphorylated Fra-1, we showed that PKCθ-induced phosphorylation of Fra-1 was crucial for the stimulation of MMP1 and IL6 expression. Consistently, we found a significant positive correlation between PRKCQ (coding for PKCθ) and MMP1 mRNA expression levels in human breast cancer samples. PKCθ-induced phosphorylations, in part at T217 and T227 residues, strongly and specifically increased Fra-1 transcriptional activity through the stimulation of Fra-1 transactivation domain, without affecting JUN factors. More importantly, these phosphorylations were required for Fra-1-induced migration of breast cancer cells and phosphorylated Fra-1 expression was enriched at the invasion front of human breast tumors. Taken together, our findings indicate that PKCθ-induced phosphorylation could be important for the function of Fra-1 in cancer progression.