Project description:We demonstrate that regorafenib treatment suppresses multiple canonical pathways and transcriptions factors We used microarrays to analyze the potential mechanism by which regorafenib regulates gene expression.
Project description:The aim of the study was to characterize the transcriptional profiles of two cholangiocarcinoma cell lines (HuCCT1 and Huh28) after a treatment with Transforming Growth Factor beta (TGF-beta).
Project description:Colorectal cancer (CRC) remains the third leading cause of cancer-related deaths worldwide, with its incidence continuing to rise. Regorafenib, a multi-kinase inhibitor approved as a palliative treatment, extends survival in patients with metastatic CRC (mCRC) who have failed standard therapies. However, the clinical benefit of regorafenib is limited to a subset of patients, is typically short-lived, and is often accompanied by significant toxicity. The mechanisms by which CRC cells develop resistance to regorafenib remain poorly understood.In this study, we investigated resistance mechanisms to regorafenib using a preclinical mouse colon organoid model. Transcriptomic analysis of Apc wild-type and Apc-deficient organoids treated with regorafenib revealed an upregulation of epithelial-to-mesenchymal transition (EMT), accompanied by alterations in the secretome and increased activation of phosphorylated Erk1/2. Notably, co-treatment with an autophagy inhibitor suppressed regorafenib-induced EMT and its associated secretory phenotype, resulting in reduced cell proliferation and enhanced apoptosis in mouse organoids. The effectiveness of this drug combination was further supported by cell viability assays in human CRC cell lines. In contrast, primary mouse colon fibroblasts displayed greater resistance to both single-agent and combination regorafenib treatments. In summary, our findings using a preclinical organoid model suggest that autophagy inhibition may offer a promising strategy to mitigate chemoresistance and reduce toxicity associated with regorafenib treatment in mCRC patients.
Project description:Developing effective model systems to evaluate new potential chemotherapeutic reagents is critical. Three-dimensional cell cultures, such as spheroids, aid in bridging the gap between commonly used monolayer cell cultures and significantly more complex and expensive animal models. Spheroid model systems contain pathophysiological and chemical gradients similar to an in vivo tumor, which ultimately form distinct cellular subpopulations. In the spatial SILAC model, labels are pulsed into the spheroid at discrete time windows during development. These media pulses result in labeled proteins in distinct regions of the spheroid, which allows for tracing of quantitative proteomic changes to be correlated to different cellular subregions. In this study, we use the Spatial SILAC model to evaluate the proteomic response of a colon carcinoma spheroid to the multikinase inhibitor Regorafenib. We determined regorafenib to be an effective kinase inhibitor which significantly altered whole spheroid proliferation and resulted in increased apoptosis when the signal was averaged for all cells in the culture. However, when regorafenib treatment is more closely examined among the cellular subpopulations, drastic differences are observed for how the drug impacted the proteome of the necrotic spheroid core and the proliferating outer regions. Whole spheroid and outer region analysis shows that regorafenib treatment inhibited critical pathways such as mTOR signaling, ERK/MAPK signaling, and colorectal cancer metastasis signaling. However, analysis of the core shows that regorafenib had an entirely different effect, including upregulation of MAPK1 and KRAS, possibly indicating drug resistance within these late apoptotic cells. Ultimately, these combinatory studies can be used to further understanding of drug metabolism is different cellular subpopulations and provide valuable information to ultimately improve the accuracy of therapeutic testing.
Project description:Regorafenib increased M1/M2 ratio of BMDMs polarization and proliferation/activation of co-cultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream CREB-KLF4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells, whereas macrophage deletion negated regorafenib’s antitumor effects. Synergistic antitumor efficacy between low-dose regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.
Project description:To evaluate the differential gene expression contributing to the efficacy of the combination treatment of JAK/HDAC inhibitor with regorafenib, compared to single drug treatment, we performed the RNA-sequencing
Project description:Low-dose regorafenib (5 mg/kg/day, corresponding to about half of human clinical dosage) inhibited tumor growth and angiogenesis in vivo similarly to DC-101 (anti-VEGFR antibody) but produced higher T cell activation and M1 macrophage polarization, Regorafenib increased M1/M2 ratio of BMDMs polarization and proliferation/activation of co-cultured T cells in vitro, indicating angiogenesis-independent immunomodulatory effects. Suppression of p38 kinase phosphorylation and downstream CREB-KLF4 activity in BMDMs by regorafenib reversed M2 polarization. Regorafenib enhanced antitumor efficacy of adoptively transferred antigen-specific T cells, whereas macrophage deletion negated regorafenib’s antitumor effects. Synergistic antitumor efficacy between low-dose regorafenib and anti-PD1 was associated with multiple immune-related pathways in the tumor microenvironment.
Project description:There is an unmet need to develop novel, effective medical therapies for cholangiocarcinoma (CCA). The Hippo pathway effector, YAP, is oncogenic in cholangiocarcinoma; however, attempts to therapeutically target YAP in vivo have been unsuccessful thus far. Recently, we described a novel role for the Src-family kinase LCK in activating YAP through tyrosine phosphorylation. This led to the hypothesis that LCK may be a viable therapeutic target in cholangiocarcinoma to regulate YAP activity. NTRC 0652-0 is a novel tyrosine kinase inhibitor with specificity for LCK. NTRC 0652-0 demonstrated selectivity for LCK inhibition in vitro and in cholangiocarcinoma cells. NTRC 0652-0 treatment led to decreased tyrosine phosphorylation of YAP, inhibition of its activity and apoptotic cell death in cholangiocarcinoma cell lines, associated with inhibition of MCL1 expression - consider rephrasing this last part to make the sentence read better. The extent of sensitivity of patient-derived organoids to NTRC 0652-0 correlated with basal YAP tyrosine phosphorylation and suppression of YAP co-transcriptional activity following drug treatment. In a patient-derived xenograft CCA model bearing an FGFR2 fusion, daily oral treatment with NTRC 0652-0 produced stable tumor drug levels, acceptable toxicity, and significantly decreased tumor growth. Overall, a novel LCK inhibitor, NTRC 0652-0, inhibited YAP signaling and demonstrated preclinical efficacy in CCA cell-lines, organoids, and patient derived xenograft models.