Tetrahydromagnolol targets TRIM38 to mediate PANoptosis in cancer cells and has the potential for synergistic cancer therapy

Loss of the Ras Regulator RASAL1: Another Route to Ras Activation in Colorectal Cancer

K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab. In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors. We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen-activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology. We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers.

Simple SummaryAF8c, a lapatinib hybrid quinazoline-based EGFR/HER2 inhibitor, was chosen to scrutinize its antiproliferative activity in colorectal cancer (CRC) cells. We found that AF8cinduced apoptosis in CRC cells via diverse mechanisms. In addition to inhibiting the phosphorylation of the ErbB family, AF8c increased the mRNA and protein levels of death receptor 5 (DR5) in vitro and in vivo. In addition, AF8c upregulated several ER stress proteins and the redox-sensitive nuclear respiratory factor 2 alpha subunit (Nrf2) in a p53-dependent manner. We also found that the AF8c-induced increase in the levels of Nrf2, DR5, and apoptosis was diminished by p53 downregulation or knockdown. Furthermore, AF8c showed higher antiproliferative activity than lapatinib in the CRC mouse model in vivo. Therefore, our results suggest AF8c as a highly effective polypharmacological small molecule with an encouraging safety profile, both in vitro and in vivo, for further evaluation as a treatment of CRC.Our team has previously reported a series of quinazoline-based lapatinib hybrids as potent kinase-targeting anticancer agents. Among them, AF8c showed a relatively safe profile in colorectal cancer (CRC) cells. In this study, we delineate a novel anticancer activity of AF8c in CRC cells. AF8c mediated p53-dependent apoptosis of CRC cells via the generation of endoplasmic reticulum (ER) stress and reactive oxygen species (ROS), as well as activation of nuclear respiratory factor 2 alpha subunit (Nrf2) and death receptor 5 (DR5), among others. The silencing of DR5 attenuated the expression levels of Nrf2 and partially inhibited AF8c-induced apoptosis. Additionally, upregulation of Nrf2 by AF8c evoked apoptosis through a decrease in antioxidant levels. Treatment of a CRC mice model with AF8c also resulted in the upregulation of DR5, Nrf2, and CHOP proteins, subsequently leading to a significant decrease in tumor burden. In comparison with lapatinib, AF8c showed higher cellular antiproliferative activity at the tested concentrations in CRC cells and synergized TRAIL effects in CRC cells. Overall, our results suggest that AF8c-induced apoptosis may be associated with DR5/Nrf2 activation through ER stress and ROS generation in CRC cells. These findings indicate that AF8c represents a promising polypharmacological molecule for the treatment of human CRC.

Details Unfold: The Endoplasmic Reticulum Stress Response in Intestinal Inflammation and Cancer

Phase II Trial of Capecitabine/Irinotecan and Capecitabine/Oxaliplatin in Advanced Gastrointestinal Cancers

<div>Abstract<p><b>Purpose:</b><i>K-Ras</i> somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong <i>in vivo</i> activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab.</p><p><b>Experimental Design:</b> In the present study, we investigated, both <i>in vitro</i> and <i>in vivo</i>, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring <i>K-Ras</i> mutations and resistance to EGFR inhibitors.</p><p><b>Results:</b> We showed that IMO was able to significantly restore the sensitivity of <i>K-Ras</i> mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen—activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. <i>In vivo</i>, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using <i>K-Ras</i> mutant colorectal cancer cell models obtained through homologous recombination technology.</p><p><b>Conclusions:</b> We showed that IMO markedly inhibits growth of <i>K-Ras</i> mutant colon and pancreatic cancers <i>in vitro</i> and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for <i>K-Ras</i> wild-type as well for <i>K-Ras</i> mutant, cetuximab-resistant colorectal and pancreatic cancers. <i>Clin Cancer Res; 17(20); 6531–41. ©2011 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b><i>K-Ras</i> somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong <i>in vivo</i> activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab.</p><p><b>Experimental Design:</b> In the present study, we investigated, both <i>in vitro</i> and <i>in vivo</i>, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring <i>K-Ras</i> mutations and resistance to EGFR inhibitors.</p><p><b>Results:</b> We showed that IMO was able to significantly restore the sensitivity of <i>K-Ras</i> mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen—activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. <i>In vivo</i>, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using <i>K-Ras</i> mutant colorectal cancer cell models obtained through homologous recombination technology.</p><p><b>Conclusions:</b> We showed that IMO markedly inhibits growth of <i>K-Ras</i> mutant colon and pancreatic cancers <i>in vitro</i> and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for <i>K-Ras</i> wild-type as well for <i>K-Ras</i> mutant, cetuximab-resistant colorectal and pancreatic cancers. <i>Clin Cancer Res; 17(20); 6531–41. ©2011 AACR</i>.</p></div>

3585 Background: Colorectal cancer (CRC) is driven by genetic alterations that result in constitutive activation of oncogenic transcription factors (eg β-catenin, MYC) and of mitogenic signaling and cell cycle progression (driven by oncogenic mutations in KRAS and BRAF). CDK7 is a key regulator of transcription, through phosphorylation of the CTD domain of RNA Polymerase II, and of cell cycle progression, through phosphorylation of the cell cycle kinases CDK1, 2, 4, and 6. This dual role of CDK7 suggests inhibitors of CDK7 may be effective in the treatment of CRC. SY-5609 is an oral, noncovalent, potent and highly selective CDK7 inhibitor in phase 1 clinical development for patients with advanced solid tumors including CRC (NCT04247126). Here we report on the activity of SY-5609 in patient-derived xenograft (PDX) models of CRC. Methods: SY-5609 was administered once daily (QD) by oral gavage for 21 days (end of treatment, EOT) to mice bearing PDX models of CRC. The relationship between SY-5609 dose, pharmacodynamic (PD) changes in xenograft tissue, tumor growth inhibition (TGI), and mouse body weight (BW) was evaluated across a range of doses. SY-5609 TGI activity was also evaluated at sub-maximum-tolerated-dose levels across a panel of 30 independent CRC models including BRAF-, KRAS-, and non-BRAF/KRAS-mutant (wild type) models (n = 10 per group). Results: SY-5609 induced dose-dependent TGI in BRAF-mutant CRC PDX tumors, with tumor regressions observed at well tolerated doses (no BW loss at EOT), and no tumor regrowth for 2+ weeks after treatment was discontinued. Dose-dependent TGI was associated with dose-dependent PD changes in PDX tumor tissue. Across 30 PDX models, SY-5609 at well-tolerated doses (average BW loss of 0% at EOT across all models) induced ≥50% TGI in 67% (20/30) of models. Deep responses (≥90% TGI or regressions) were observed in 23% (7/30) of models, with enrichment for deep responses in BRAF mutant models (50%, 5/10) relative to KRAS mutant (10%, 1/10), and wild type (10%, 1/10) models. Conclusions: Daily oral dosing of the CDK7 inhibitor SY-5609 induces robust TGI, including regressions, in CRC PDX models at well-tolerated doses. Dose-dependent TGI is associated with dose-dependent PD changes in CRC PDX tumor tissue. These results highlight the therapeutic potential of SY-5609 in CRC and support the evaluation of SY-5609 in CRC patients in early phase clinical trials. SY-5609 is in phase 1 clinical development for patients with advanced solid tumors including CRC (NCT04247126).

Despite advances in screening procedures and the use of adjuvant therapy, approximately 50% of patients with colorectal cancer eventually will develop metastatic disease. Long-term disease-free survival can be achieved in 25% to 40% of selected patients who undergo resection of liver or lung metastases. For all other patients, treatment is palliative. For decades, 5-fluorouracil was the only available drug for colorectal cancer; hence, numerous trials were performed that used various administration schedules and modulating agents to improve therapeutic efficacy. The addition of leucovorin to 5-FU improves response but not survival. Infusion schedules alter the toxicity profile but have a negligible impact on survival. Irinotecan was the first new drug to demonstrate activity in colorectal cancer. It was used initially in the second-line setting, where it was shown to improve quality of life and survival over best supportive care or infusional 5-FU. Recently, irinotecan has been incorporated into the front-line treatment of metastatic colorectal cancer in combination with 5-FU and leucovorin; this combination improves survival by approximately 3 months. Careful patient selection and adherence to strict dose adjustments are essential to prevent significant toxicity when patients are treated on this regimen. The oral fluoropyrimidine capecitabine recently has been approved for the front-line treatment of patients with colorectal cancer who are not appropriate candidates for combination therapy. Oxaliplatin, a novel DACH (diaminocyclohexane) platinum with definite activity in colorectal cancer, is approved for this disease in Europe and is undergoing phase III clinical trials in the United States. Other drugs with potential activity in colorectal cancer include raltitrexed, pemetrexed disodium, and the epothilone analog BMS-247550 (Bristol-Myers Squibb, New York, NY). Novel cytostatics with promising activity in colorectal cancer are being evaluated in clinical trials, including epidermal growth factor receptor inhibitors, such as IMC-C225 (Imclone Systems, New York, NY) and ZD1839 (AstraZeneca, London, UK), angiogenesis inhibitors such as bevacizumab and SU5416 (Sugen, San Francisco, CA), and vaccines such as CEAVac (Titan Pharmaceuticals, San Francisco, CA). For those patients whose disease is localized to the liver, there also is an emerging role for local therapies, including cryosurgery, radiofrequency ablation, and hepatic artery infusional chemotherapy, and resection. The emergence of these new drugs and new interventional modalities has allowed physicians who treat colorectal cancer to move beyond 5-FU.

Protein disulfide isomerase inhibits endoplasmic reticulum stress response and apoptosis via its oxidoreductase activity in colorectal cancer

BackgroundThe incidence of colorectal cancer and cancer death rate are increasing every year, and the affected population is becoming younger. Traditional Chinese medicine therapy has a unique effect in prolonging survival time and improving the prognosis of patients with colorectal cancer. Oridonin has been reported to have anti-cancer effects in a variety of tumors, but the exact mechanism remains to be investigated.MethodsCell Counting Kit-8 assay (CCK8) and 5-Ethynyl-2'-deoxyuridine (EdU) staining assay, Tranwell, and Wound healing assays were performed to measure cell proliferation, invasion, and migration capacities, respectively. The protein and mRNA expression levels of various molecules were reflected by Western blot and Reverse Transcription quantitative Polymerase Chain Reaction (qRT-PCR). Transcription Factor 4 (TCF4) and its target genes were analyzed by Position Weight Matrices (PWMs) software and the Gene Expression Omnibus (GEO) database. Immunofluorescence (IF) was performed to visualize the expression and position of Endoplasmic Reticulum (ER) stress biomarkers. The morphology of the ER was demonstrated by the ER tracker-red. Reactive Oxygen Species (ROS) levels were measured using a flow cytometer (FCM) or fluorescent staining. Calcium ion (Ca2+) concentration was quantified by Fluo-3 AM staining. Athymic nude mice were modeled with subcutaneous xenografts.ResultsOridonin inhibited the proliferation, invasion, and migration of colorectal cancer, and this effect was weakened in a concentration-dependent manner by ER stress inhibitors. In addition, oridonin-induced colorectal tumor cells showed increased expression of ER stress biomarkers, loose morphology of ER, increased vesicles, and irregular shape. TCF4 was identified as a regulator of ER stress by PWMs software and GEO survival analysis. In vitro and in vivo experiments confirmed that TCF4 inhibited ER stress, reduced ROS production, and maintained Ca2+ homeostasis. In addition, oridonin also activated TP53 and inhibited TCF4 transactivation, further exacerbating the elevated ROS levels and calcium ion release in tumor cells and inhibiting tumorigenesis in colorectal cancer cells in vivo.ConclusionsOridonin upregulated TP53, inhibited TCF4 transactivation, and induced ER stress dysregulation in tumor cells, promoting colorectal cancer cell death. Therefore, TCF4 may be one of the important nodes for tumor cells to regulate ER stress and maintain protein synthesis homeostasis. And the inhibition of the TP53/TCF4 axis plays a key role in the anti-cancer effects of oridonin.

Colorectal adenocarcinoma (COAD) is a significant cause of cancer-related mortality worldwide, necessitating the identification of novel therapeutic targets and treatments. This research aimed to investigate the role of ARL3 in COAD progression and to explore the effects of Piperine on ARL3 expression, cell proliferation, epithelial–mesenchymal transition (EMT), and endoplasmic reticulum (ER) stress. Bioinformatics analysis of The Cancer Genome Atlas (TCGA)–COAD, GSE39582, and GSE44861 datasets assessed ARL3 expression levels. Immunohistochemical data from the Human Protein Atlas (HPA) database confirmed ARL3 overexpression in COAD. The association of ARL3 with COAD clinical parameters and prognosis was also examined. COAD cells were treated with Piperine, and in vitro assays evaluated cell proliferation, apoptosis, EMT marker expression, and ER stress (ERS) responses. ARL3 overexpression in COAD correlated with poor prognosis and varied across pathological stages. Piperine treatment inhibited COAD cell proliferation in a concentration- and time-dependent manner, as indicated by reduced Ki-67 levels and decreased colony-forming ability. Piperine induced S-phase cell cycle arrest and facilitated apoptosis in COAD cells, evidenced by changes in Bax, Bcl-2, cleaved caspase-3, and cleaved Poly (ADP-ribose) polymerase (PARP) levels. Moreover, Piperine downregulated ARL3 expression in COAD cells, thereby suppressing transforming growth factor beta (TGF-β)-induced EMT. Additionally, Piperine attenuated the ARL3-mediated ER stress response, significantly reducing binding immunoglobulin protein (BiP), inositol-requiring enzyme 1 alpha (p-IRE1α), activating transcription factor 6 (ATF6), and C/EBP homologous protein (CHOP) levels. Piperine exerts anti-cancer effects in COAD by modulating ARL3 expression, disrupting cell cycle progression, inhibiting the EMT pathway, and regulating ERS. These findings suggest that Piperine holds promise as a therapeutic agent for COAD through its targeting of ARL3.

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality worldwide. Natural compounds with anticancer potential, such as tomentosin, a sesquiterpene lactone derived from Inula viscosa, are under investigation as alternative therapeutic agents. However, its potential effects on CRC remain unexplored. This study aimed to evaluate the anticancer potential of tomentosin in CRC cells and elucidate its underlying molecular mechanisms. HCT 116 and HT- 29 cells were treated with tomentosin, and its effects on cell viability, colony formation, invasion, apoptosis, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, autophagy, and endoplasmic reticulum (ER) stress were evaluated. Various assays, including XTT, colony formation, and Matrigel invasion assays, were used to assess cell viability, proliferation, and invasion. Tomentosin markedly reduced cell viability and colony formation in a dose-dependent manner. It suppressed invasion and induced apoptosis, as evidenced by an increased apoptotic index and upregulation of CASP3, CASP7, CASP8, CASP9, and BAX. Tomentosin disrupted MMP and elevated ROS levels, contributing to apoptotic signaling. Autophagic activity was significantly upregulated, with increased expression of BECLIN1, ATG5, ATG7, and MAP1LC3 A. ER stress markers GRP78, ATF6, CHOP, and XBP1 were also upregulated, suggesting a role in cell death. Tomentosin has anticancer effects in CRC cells by inducing apoptosis, modulating autophagy, and triggering ER stress. These findings underscore tomentosin’s potential as a novel therapeutic candidate for CRC, warranting further in vivo and clinical investigations.

BackgroundRecently, colorectal cancer has become a more common type of tumor in the world. Research has reported that several kinds of single compounds of Chinese herbs have shown anti-tumor activity in colorectal cancer. Aloe-emodin (AE), a natural compound extract from Aloe Vera, has been discovered to suppress cell proliferation and accelerate apoptosis in a variety of tumor cells. Whether AE exerts an effect on colorectal cancer cells has not yet been investigated.Material/MethodsIn this study, we examined the impact of AE on SW620 and HT29 colorectal cancer cell lines. After treatment with various concentrations of AE (10, 20, and 40 μM), cell proliferation, cell apoptosis, reactive oxygen species (ROS) generation, cytosolic calcium level, and related gene expression were analyzed.ResultsOur results indicated that AE suppressed cell viability and induced cell apoptosis in SW620 and HT29 cell lines. Furthermore, both cell lines when exposed to AE generated ROS, which induces endoplasmic reticulum (ER) stress. We then detected the expression of ER stress-related proteins and cytosolic calcium levels. We found that cells exposure to AE had upregulation of unfolded protein response (UPR) proteins like glucose-related protein 78 (GRP78), phosphorylated protein kinase R (PKR)-like ER kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α), and transcription factor C/EBP homologous protein (CHOP). Meanwhile, we detected an increased cytosolic calcium content followed by the upregulation of the calpain-1, calpain-2 and caspase-12. CHOP and caspase-12 are important regulatory factors leading to cell apoptosis.ConclusionsAE might serve as a candidate in the treatment of colorectal cancer through inducing ER stress-dependent apoptosis.

Chronic inflammation and oncogenic pathway activation are key-contributing factors in colorectal cancer pathogenesis. However, colorectal intrinsic mechanisms linking these two factors in cancer development are poorly defined. Here, we show that intestinal epithelial cell (IEC)-specific deletion of Dot1l histone methyltransferase (Dot1lΔIEC ) reduced H3K79 dimethylation (H3K79me2) in IECs and inhibited intestinal tumor formation in ApcMin - and AOM-DSS-induced colorectal cancer models. IEC-Dot1l abrogation was accompanied by alleviative colorectal inflammation and reduced Wnt/β-catenin signaling activation. Mechanistically, Dot1l deficiency resulted in an increase in Foxp3+RORϒ+ regulatory T (Treg) cells and a decrease in inflammatory Th17 and Th22 cells, thereby reducing local inflammation in the intestinal tumor microenvironment. Furthermore, Dot1l deficiency caused a reduction of H3K79me2 occupancies in the promoters of the Wnt/β-catenin signaling genes, thereby diminishing Wnt/β-catenin oncogenic signaling pathway activation in colorectal cancer cells. Clinically, high levels of tumor H3K79me2 were detected in patients with colorectal carcinomas as compared to adenomas, and negatively correlated with RORϒ+FOXP3+ Treg cells. Altogether, we conclude that DOT1L is an intrinsic molecular node connecting chronic immune activation and oncogenic signaling pathways in colorectal cancer. Our work suggests that targeting the DOT1L pathway may control colorectal carcinogenesis. Significance: IEC-intrinsic DOT1L controls T cell subset balance and key oncogenic pathway activation, impacting colorectal carcinogenesis.