FOXK1 drives colorectal cancer progression by transcriptionally activating GRB2.

The role of JAK/STAT signaling pathway and TNF-α crosstalk in human colorectal cancer

The intestinal epithelium acts as a physical barrier that separates the intestinal microbiota from the host and is critical for preserving intestinal homeostasis. The barrier is formed by tightly linked intestinal epithelial cells (IECs) (i.e. enterocytes, goblet cells, neuroendocrine cells, tuft cells, Paneth cells, and M cells), which constantly self-renew and shed. IECs also communicate with microbiota, coordinate innate and adaptive effector cell functions. In this review, we summarize the signaling pathways contributing to intestinal cell fates and homeostasis functions. We focus especially on intestinal stem cell proliferation, cell junction formation, remodelling, hypoxia, the impact of intestinal microbiota, the immune system, inflammation, and metabolism. Recognizing the critical role of KRAS mutants in colorectal cancer, we highlight the connections of KRAS signaling pathways in coordinating these functions. Furthermore, we review the impact of KRAS colorectal cancer mutants on pathway rewiring associated with disruption and dysfunction of the normal intestinal homeostasis. Given that KRAS is still considered undruggable and the development of treatments that directly target KRAS are unlikely, we discuss the suitability of targeting pathways downstream of KRAS as well as alterations of cell extrinsic/microenvironmental factors as possible targets for modulating signaling pathways in colorectal cancer.2BMDrgCx2PNRo1_MkpS-1DVideo

Background: Colorectal cancer (CRC) is a significant global public health concern. It remains one of the leading causes of cancer-related deaths among Puerto Rican Hispanics (PRH), with 60-70% of cases diagnosed at advanced stages (III/IV). This highlights the urgent need for novel diagnostic biomarkers for early-stage CRC detection while monitoring those in advanced stages, which is crucial for understanding progression and guiding therapy. The Wnt signaling pathway plays a vital role in cell proliferation, differentiation, apoptosis, and cancer progression. This study aims to identify stage-specific biomarkers linked to the Wnt signaling pathway and the molecular mechanisms associated with CRC disease progression. Methods: A retrospective case-case study was conducted on 30 tissue samples from PRH recruited through the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR). Transcriptomic analysis was performed in tissue samples from early (n=7) and advanced CRC stages (n=8) compared to their respective adjacent mucosal tissue as controls (adjusted p-value < 0.05). We used R statistical software (package DESeq2) for gene expression analysis between groups stratified by CRC tumor stage. Protein-protein interaction networks were generated using the STRING database to explore functional associations. Specific Wnt signaling markers, target genes, and molecular processes associated with each CRC stage will be validated using Quantinova PCR. Results: Functional interaction networks analysis (≥ 5 genes) revealed 10 Wnt signaling-associated genes uniquely deregulated in early-stage CRC and 10 in advanced-stage CRC. Differentially expressed genes in early stages included WNT1, DKK1, and DKK2, while WNT3 was exclusive to advanced stages. Additional Wnt-related genes, such as WNT2, WNT7B, and WNT5, were differentially expressed in both early and advanced stages. These findings highlight distinct patterns of Wnt signaling pathway involvement across CRC stages. Conclusion: Differential gene expression analysis of early and advanced CRC stages revealed significant deregulation of Wnt signaling pathway genes, emphasizing their potential as stage-specific diagnostic biomarkers and therapeutic targets. Future studies will focus on the modulation of these genes to study the expression and molecular mechanisms that can contribute to CRC disease progression. Understanding these molecular mechanisms is of utmost importance, as it will facilitate the development of more effective, targeted strategies for CRC diagnosis and treatment, improving outcomes for at-risk populations particularly PRH. Citation Format: Elba V. Caraballo, Camille Zenón-Meléndez, Josué Pérez-Santiago, Sheila López-Acevedo, Marcia R. Cruz-Correa. Stage-specific deregulation of the Wnt signaling pathway in colorectal cancer among Puerto Rican Hispanics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3274.

Tumor specific alterations in miRNA provide ideal biomarkers for early diagnosis of colorectal cancer(CRC). Methylation changes play an important role in the miRNA regulation mechanism involved in tumorigenesis. Therefore, it is important to find miRNA methylation markers related to tumor proliferation and metastasis, and to explore its key regulatory mechanisms. Previous integrated bioinformatics analysis suggested that reduced expression and hypermethation of miR-146a have detected in majority of CRC. However, its biological behavior in CRC remains unclear. This article aims to explore its epigenetic regulation and mechanism in the development of CRC. We collected peripheral blood and tissues from 60 patients with advanced primary CRC, analyzed their clinicopathological characteristics. The mRNA and methylation difference was detected by qPCR and MSP respectively, and the significance in the diagnosis of CRC was analyzed. The research was further confirmed in SW480, SW620, HCT116 and HT29 CRC cell lines, as well as CRC animal models treated with demethylated drugs–5-aza-miR2-oxyeytidineine(5-AZA) and various gene transfection, in order to explore the key downstream regulatory network of miR-146a methylation changes and the effect of methylation inhibitors in the development of CRC. We found that the percentage of down-regulated and methylation of miR-146a in both tissues and plasmas of CRC was significantly higher than that of normal samples, and the expression of miR-146a could be recovered after demethylation treatment in cancer, which was associated with clinical stage. miR-146a methylation was significantly correlated with grading, but not gender, tumor location, histological type or TNM, it may be used as a predictor of poor prognosis. Diagnostic evaluation of miR-146a methylation in tissue and plasma was evaluated using ROC curve, area under regional curve, which indicated that miR-146a methylation could be used as a biomarker for the diagnosis of CRC. Fluorescein report system showed that miR-146a targeted VASN protein, and while miR-146a was overexpressed after transfection into CRC cells, the expression levels of VASN, TGF-β, VEGF-C, MMP9, and Vimentin were significantly reduced, while the expression of TIMP1 and Ecadherin were up-regulated. Additionally, the miR-146a mRNA in CRC tissues was also negatively correlated with the expression of VASN. VASN Silencing down-regulated TGF-β expression, accompanied by a decrease of MMP9. At the same time, after 5-aza demethylation or siVASN, proliferation activity of CRC cells decreased, accompanied by varying degrees of decline in membrane penetrating and migration ability, suggesting that miR-146a may regulate the proliferation and metastasis by regulating VASN/TGF-β relatived pathway. In vivo animal models, it was further confirmed that overexpressed miR-146a was consistent with the effect of 5-aza intervention or siVASN, which could significantly inhibit the occurrence of liver metastasis of CRC. Demethylated drugs treatment or VASN knockdown also significantly inhibited tumor growth in nude mice with CRC. miR-146a was a potential epigenetic silencing tumor suppressor in CRC, and the methylation of miR-146a may play an important role in the diagnosis and prediction of tumor progression in CRC as a new tumor marker. Citation Format: Weiwei Tang, Dan Zhou, Hanxiang An, Jiapeng Kang, Mingquan Cai, Ru Zeng, Jing Song, Bin Hu, Jiabian Lian, Qin Lin, Lilin Chen, Feng Ye. miR-146a methylation regulates proliferation and metastasis by targeted activation of VASN/TGF-beta signaling pathway in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2443.

CRABP2 promotes peritoneal metastasis in CRC through TGF-β/Smad-mediated EMT signaling and invadopodia formation.

Simple SummaryColorectal cancer (CRC) is the fourth most common cancer and the second most common cause of cancer-related deaths globally. Rhein is a natural anthraquinone extract from rhubarb, which exhibits potent anticancer activity in various cancers. In this study, we show that rhein significantly inhibited the growth, migration, and invasion of CRC cells by directly binding to mTOR and inhibiting the mTOR signaling pathway. Rhein promotes mTOR degradation through the ubiquitin–proteasome pathway. In addition, rhein significantly suppressed tumor growth in a xenograft mouse model without obvious toxicity. Our results indicate that rhein is a promising anticancer agent that may be useful for the prevention and treatment of CRC.Colorectal cancer (CRC) is one of the leading causes of mortality and morbidity in the world. Rhein has demonstrated therapeutic effects in various cancer models. However, its effects and underlying mechanisms of action in CRC remain poorly understood. We investigated the potential anticancer activity and underlying mechanisms of rhein in CRC in vitro and in vivo. Cell viability and anchorage-independent colony formation assays were performed to examine the antigrowth effects of rhein on CRC cells. Wound-healing and Transwell assays were conducted to assess cell migration and invasion capacity. Cell cycle and apoptosis were investigated by flow cytometry and verified by immunoblotting. A tissue microarray was used to detect mTOR expression in CRC patient tissues. Gene overexpression and knockdown were done to analyze the function of mTOR in CRC. The anticancer effect of rhein in vivo was assessed in a CRC xenograft mouse model. The results show that rhein significantly inhibited CRC cell growth by inducing S-phase cell cycle arrest and apoptosis. Rhein inhibited CRC cell migration and invasion through the epithelial–mesenchymal transition (EMT) process. mTOR was highly expressed in CRC cancer tissues and cells. Overexpression of mTOR promoted cell growth, migration, and invasion, whereas mTOR knockdown diminished these phenomena in CRC cells in vitro. In addition, rhein directly targeted mTOR and inhibited the mTOR signaling pathway in CRC cells. Rhein promoted mTOR degradation through the ubiquitin-proteasome pathway. Intraperitoneal administration of rhein inhibited HCT116 xenograft tumor growth through the mTOR pathway. In conclusion, rhein exerts anticancer activity in vitro and in vivo by targeting mTOR and inhibiting the mTOR signaling pathway in CRC. Our results indicate that rhein is a potent anticancer agent that may be useful for the prevention and treatment of CRC.

Background: The development of chemo-resistance against conventional chemotherapeutic drugs presents a major clinical challenge in the management of patients with colorectal cancer (CRC). Accumulating body of data in recent years have shown that active principles within various naturally-occurring dietary botanicals not only offer time-tested safety and anti-cancer efficacy, but a combination of certain compounds can overcome the elusive chemotherapeutic resistance in cancer patients. In this regard, data in recent years have shown that berberine (BBR) and oligomeric proanthocyanidins (OPCs) from the grape-seed extract have significant anti-tumorigenic properties in CRC, and a more recent study reported existence of synergism between these two natural medicines in patients with type 2 diabetes. Accordingly, herein we hypothesized that BBR and OPCs might regulate synergistically multiple oncogenic pathways to exert a superior anti-cancer activity in CRC. Methods: We performed a series of experiment in various cell lines, followed by their interrogation in patient-derived organoids (PDOs) to evaluate the synergistic anti-tumorigenic effects of BBR and OPCs in CRC cells. In addition, using genome-wide RNA sequencing analyses, we identified specific functional determinants and key targeted genes and pathways regulated by the combined therapeutic approach with these compounds. Results: To confirm our hypothesis, we first evaluated the potential of OPCs to increase the cellular uptake of BBR in CRC cells, by measuring the fluorescent signal of BBR in multiple cell lines treated individually or their combination. The synergistic anti-tumorigenic effects of BBR and OPCs were observed in various functional assays including reduced cell viability, colony formation potential, wound healing, and invasion assays. Furthermore, the combined treatment with these compounds potentiated the cell apoptosis in an Annexin V binding assay. It was intriguing to note that we were able to successfully validate all cell culture findings in the PDO models from multiple CRC patients. Whole genome transcriptomic profiling identified oncogene MYB, including in PI3-AKT signaling pathway as one of the key pathways that was critically involved in mediating the anti-tumorigenic properties of this combined therapy. Conclusions: We for the first time demonstrate that a combined treatment with BBR and OPCs synergistically promotes the anti-tumorigenic properties in CRC. This synergism was potentially due to enhanced apoptosis and the regulation of oncogenic MYB in the PI3-Akt signaling pathway; hence, offering an attractive potential for the use of these two compounds as adjuvant treatment options in patients with CRC. Citation Format: Keisuke Okuno, Rachana Garg, Masanori Tokunaga, Yusuke Kinugasa, Ajay Goel. Synergistic antitumorigenic activity of Berberine and oligomeric proanthocyanidins through regulation of PI3-AKt signaling pathway in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1409.

Colorectal cancer is a common malignancy. NTS receptor 3 (NTSR3) is known to play an important role in several cancers. This study examined the effects of NTSR3 on cell growth and metastasis in colorectal cancer. Western blot analysis, real-time PCR, immunofluorescence staining, MTT, cell cycle assay, cell apoptosis assay, Hoechst staining, caspase-3 and caspase-9 activity assays, cell adhesion assay, wound healing assay, and a Transwell assay were used in this study. We found that NTSR3 was expressed at relatively high levels in the colorectal cancer cell lines SW620 and SW480. NTSR3 knockdown suppressed cell growth and promoted cell apoptosis. Meanwhile, the protein expression levels of cyclinD1, cyclinE1, CDK4, and p-RB were reduced, and the levels of p-P27, P15, P21, cleaved caspase-3, and cleaved caspase-9 protein were increased. Cell invasiveness and cell migration were reduced with knockdown of NTSR3. In addition, our rescue experiments demonstrated that overexpression of the siRNA-resistant alleles of NTSR3 abrogated the NTSR3-siRNA-mediated effects on cell function. Further, down-regulation of NTSR3 inactivated the PI3K-AKT and MAPK signaling pathways. Collectively, these data demonstrate that knockdown of NTSR3 inhibits cell growth and metastasis, as well as the PI3K-AKT and MAPK signaling pathways in colorectal cancer. Thus, our results indicate that NTSR3 is a potential therapeutic target for treating colorectal cancer.

Colorectal cancer (CRC) results from the accumulation of gene mutations and epigenetic alterations in colon epithelial cells, which promotes CRC formation through deregulating signaling pathways. One of the most commonly deregulated signaling pathways in CRC is the transforming growth factor β (TGF-β) pathway. Importantly, the effects of TGF-β signaling inactivation in CRC are modified by concurrent mutations in the tumor cell, and these concurrent mutations determine the ultimate biological effects of impaired TGF-β signaling in the tumor. However, many of the mutations that cooperate with the deregulated TGF-β signaling pathway in CRC remain unknown. Therefore, we sought to identify candidate driver genes that promote the formation of CRC in the setting of TGF-β signaling inactivation. We performed a forward genetic screen in mice carrying conditionally inactivated alleles of the TGF-β receptor, type II (Tgfbr2) using Sleeping Beauty (SB) transposon mediated mutagenesis. We used TAPDANCE and Gene-centric statistical methods to identify common insertion sites (CIS) and, thus, candidate tumor suppressor genes and oncogenes within the tumor genome. CIS analysis of multiple neoplasms from these mice identified many candidate Tgfbr2 cooperating genes and the Wnt/β-catenin, Hippo and MAPK pathways as the most commonly affected pathways. Importantly, the majority of candidate genes were also found to be mutated in human CRC. The SB transposon system provides an unbiased method to identify Tgfbr2 cooperating genes in mouse CRC that are functionally relevant and that may provide further insight into the pathogenesis of human CRC.

Tumor necrosis factor-induced protein-8 (TIPE) is highly expressed in colorectal cancer (CRC). Decoy receptor 3 (DcR3) is a soluble secreted protein that can antagonize Fas ligand (FasL)-induced apoptosis and promote tumorigenesis. It remains unclear whether TIPE can regulate DcR3 expression. In this study, we examined this question by analyzing the relationship between these factors in CRC. Bioinformatics and tissue microarrays were used to determine the expression of TIPE and DcR3 and their correlation in CRC. The expression of TIPE and DcR3 in colon cancer cells was detected. Plasma samples were collected from CRC patients, and DcR3 secretion was measured. Then, dual-luciferase reporter gene analysis was performed to assess the interaction between TIPE and DcR3. We exogenously altered TIPE expression and analyzed its function and influence on DcR3 secretion. Lipopolysaccharide (LPS) was used to stimulate TIPE-overexpressing HCT116 cells, and alterations in signaling pathways were detected. Additionally, inhibitors were used to confirm molecular mechanisms. We found that TIPE and DcR3 were highly expressed in CRC patients and that their expression levels were positively correlated. DcR3 was highly expressed in the plasma of cancer patients. We confirmed that TIPE and DcR3 were highly expressed in HCT116 cells. TIPE overexpression enhanced the transcriptional activity of the DcR3 promoter. TIPE activated the PI3K/AKT signaling pathway to regulate the expression of DcR3, thereby promoting cell proliferation and migration and inhibiting apoptosis. In summary, TIPE and DcR3 are highly expressed in CRC, and both proteins are associated with poor prognosis. TIPE regulates DcR3 expression by activating the PI3K/AKT signaling pathway in CRC, thus promoting cell proliferation and migration and inhibiting apoptosis. These findings may have clinical significance and promise for applications in the treatment or prognostication of CRC.

Colorectal cancer (CRC) is the third most common malignancy and one of the leading causes of cancer-related death among men worldwide. CRC is a multifactor digestive pathology, which is a huge problem faced not only by clinicians but also by researchers. Importantly, a unique feature of CRC is the dysregulation of molecular signaling pathways. To date, a series of reviews have indicated that different signaling pathways are disordered and have potential as therapeutic targets in CRC. Nevertheless, an overview of the function and interaction of multiple signaling pathways in CRC is needed. Therefore, we summarized the pathways, biological functions and important interactions involved in CRC. First, we investigated the involvement of signaling pathways, including Wnt, PI3K/Akt, Hedgehog, ErbB, RHOA, Notch, BMP, Hippo, AMPK, NF-κB, MAPK and JNK. Subsequently, we discussed the biological function of these pathways in pathophysiological aspects of CRC, such as proliferation, apoptosis and metastasis. Finally, we summarized important interactions among these pathways in CRC. We believe that the interaction of these pathways could provide new strategies for the treatment of CRC.

Convallatoxin promotes apoptosis and inhibits proliferation and angiogenesis through crosstalk between JAK2/STAT3 (T705) and mTOR/STAT3 (S727) signaling pathways in colorectal cancer

Epigenetic silencing of tumor suppressors contributes to the development and progression of colorectal cancer (CRC). We recently found that speckle-type POZ protein (SPOP) was significantly downregulated and the inactivation of SPOP promoted metastasis in CRC. This study aimed to clarify its epigenetic alteration, molecular mechanisms and clinical significance in CRC. Our results revealed that the core region of SPOP promoter was hypermethylated in CRC tissues and its methylation was correlated with poor survival. Transcription factor RXRA had a vital role in the regulation of SPOP gene. The data indicated that DNA methylation at −167 bp of the SPOP gene altered the binding affinity between transcription factor RXRA and SPOP promoter. Moreover, SPOP was found to associate with Gli2 and promoted its ubiquitination and degradation in CRC. Consequently, the expression level of Hh/Gli2 pathway-related apoptotic protein Bcl-2 was decreased and the function of resisting cell death was inhibited in CRC. It suggests that methylation status of SPOP promoter can be used as a novel epigenetic biomarker and a therapeutic target in CRC.

Intersection of Transforming Growth Factor-β and Wnt Signaling Pathways in Colorectal Cancer and Metastasis

Genetic alterations of the TGF-β signaling pathway in colorectal cancer cell lines: A novel mutation in Smad3 associated with the inactivation of TGF-β-induced transcriptional activation