Expression Profiling of EMT Transcription Factors in Epithelial Cancer vs. Colon Cancer from a Clini-cal Dataset (Snail, Slug, Zeb-1, Twist)

Abstract

Colorectal cancer (CRC) is the second most common cancer and a leading cause of cancer-related deaths worldwide. Epithelial-mesenchymal transition (EMT) is a biological process that enables a polarized epithelial cell to go through a variety of biochemical changes and adopt the phenotype of a mesenchymal cell, which includes increased migratory ability and invasive-ness. Here, we aim to explore the role of a comprehensive analysis of the dynamic regulation of EMT transcription factors Slug, Snail, Twist, and Zeb1 in CRC. By examining expression patterns across different tumor stages, age groups, and genders, the research elucidates the stage-dependent and demographic-specific modulation of EMT drivers. Findings reveal that key EMT regulators such as Slug and Snail are predominantly active during early CRC stages, contributing to tumor initiation and local invasion. At the same time, Twist and Zeb1 become more prominent during advanced stages, facilitating metastasis and tumor progression. Notably, gender differences influence the expression of these factors, with females exhibiting higher levels of Slug and Twist. The study underscores the potential of these transcription factors as stage- and patient-specific biomarkers and thera-peutic targets, emphasizing the importance of personalized strategies in CRC management. Future investigations aimed at func-tional validation and clinical trials could pave the way for stage-specific EMT-targeted interventions, ultimately improving prognosis and treatment efficacy in CRC.

Introduction

Epithelial-Mesenchymal Transition (EMT) is a biological process in which epithelial cells lose their cell polarity and adhesion and acquire mesenchymal traits, including increased motility and invasiveness. EMT is critical in:

• Embryonic development

• Wound healing

• Cancer progression, especially metastasis

During EMT:

• Epithelial markers (e.g., E-cadherin, ZO-1, cytokeratin) are downregulated

• Mesenchymal markers (e.g., N-cadherin, vimentin, fibronectin) are upregulated

• Cell junctions (TJs, AJs, desmosomes) are disassembled

2. Key EMT Transcription Factors (TFs)

EMT is regulated by several transcription factors, including:

A. Snail (SNAI1)

• Master EMT regulator

• Represses E-cadherin by binding to E-boxes

• Promotes mesenchymal markers (e.g., Vimentin)

• Induced by TGF-β, Wnt, Notch signaling

• Associated with poor prognosis, metastasis, chemoresistance

B. Slug (SNAI2)

• Similar to Snail

• Strongly represses E-cadherin

• Promotes invasion, EMT in CRC, melanoma, lung, renal, and gastric cancers

• Overexpression linked to advanced disease and shorter survival

C. Zeb1

• Zinc-finger homeodomain TF

• Binds E-boxes to repress epithelial genes (e.g., CDH1/E-cadherin)

• Regulates genes involved in chemoresistance, immune evasion, and stemness

• Associated with poor prognosis in CRC, lung cancer, gastric cancer, kidney cancer

D. Twist1

• bHLH TF important in mesoderm development

• Promotes EMT, chemoresistance, metastasis in CRC, lung, and gastric cancers

• Linked to genomic instability, cancer stemness, and DNA damage tolerance

3. EMT in Colorectal Cancer (CRC)

• EMT plays a key role in CRC progression from adenoma to metastatic carcinoma

• Snail1, Slug, Zeb1, and Twist contribute to tumor invasion, stemness, and chemoresistance

• Snail1 and Slug are active early, while Twist and Zeb1 become dominant in later/metastatic stages

• Snail and Slug expression is inversely correlated with E-cadherin

• EMT activity is higher in females and younger patients

4. EMT TFs in Other Cancers

• Melanoma: Snail1 in fibroblasts promotes tumor growth and immunosuppression

• Lung cancer: Snail and Slug enhance invasion and resistance to EGFR-targeted therapy

• Renal cell carcinoma (RCC): Snail and Slug are potential prognostic markers

• Gastric cancer: Slug and Twist associated with metastasis and poor survival

5. Mechanistic Insights

• EMT TFs recruit co-repressors (e.g., CtBP, HDAC3) and co-activators (Smad, p300) to modify chromatin

• EMT is regulated post-transcriptionally and post-translationally

• Zeb1 and non-coding RNAs (e.g., Zeb1-AS1, LINC01559) regulate gene expression through ceRNA networks

• Zeb1 can induce PD-L1 expression, promoting immune evasion

6. Bioinformatics & Data Analysis Methods

• Expression data for EMT TFs was downloaded from the Human Protein Atlas (HPA)

• Normal vs. cancerous tissues, cancer stages, and gender-specific datasets were analyzed

• FPKM values were normalized for cross-sample comparison

• Statistical analyses identified key EMT TFs with significant roles across stages and genders in CRC

7. Key Observations

• Slug and Snail: Prominent in early CRC, especially in females and younger patients

• Twist and Zeb1: Upregulated in advanced stages and metastasis

• EMT-related expression is stage-dependent and gender-influenced

• Targeting EMT TFs may offer therapeutic potential in stage-specific and personalized CRC treatments

Conclusion

The comprehensive evaluation of EMT transcription factor expression in CRC across stages, age groups, and genders highlights a coordinated and dynamic regulation of EMT throughout disease progression. In the early stages (Stage I and II) of CRC, Slug and Snail emerge as key drivers of EMT initiation. Their elevated expression, particularly in younger patients and female individuals, suggests that these factors are critical in estab-lishing early epithelial-to-mesenchymal plasticity, facilitating tumor cell detachment, local invasion, and early stromal interactions. This phase represents the transition from benign epithelial behavior to an invasive phenotype, setting the foundation for further tumor progression. As the disease advances into Stage III and IV, there is a noticeable shift in the EMT landscape, with Twist and Zeb1 taking on more dominant roles. Twist expression becomes increasingly prominent in later stages, especially in female patients, indicating its contribution to sustained EMT, migration, and possibly distant metastasis. Zeb1, although consistently lower in CRC compared to normal tissues, shows a relative increase in advanced stages, suggesting a reactivation or compensation mechanism that supports prolonged mesenchymal traits and survival advantages in hostile microenvironments. Notably, gender-based differences are evident throughout: females consistently show higher expression of Slug and Twist, pointing to a sex-specific modulation of EMT that could influence disease aggressiveness, therapeutic response, or prognosis. Age-wise, the variation in expression is subtle but informative younger CRC patients tend to express higher levels of Snail and Twist, hinting at a more aggressive EMT phenotype in early-onset CRC cases. In contrast, Slug becomes more prominent with increasing age, peaking in older individuals, potentially reflecting age-associated epigenetic or inflammatory influences on EMT regulation. Taken together, these findings underscore a stage-dependent transition in EMT drivers with Slug and Snail initiating EMT in early CRC and Twist and Zeb1 contributing to progression and metastasis in later stages. The interplay of age and gender further refines this regulatory pattern, offering insight into how demographic factors may influence EMT dynamics and CRC behavior. These observations not only enhance our understanding of EMT in CRC but also suggest that targeted interventions could be more effective if tailored to stage- and patient-specific EMT profiles.

References

[1] D. M. Gonzalez and D. Medici, “Signaling mechanisms of the epithelial-mesenchymal transition,” 2014. doi: 10.1126/scisignal.2005189. [2] Heidi Kurn, Histology, Epithelial Cell. 2023. Accessed: Feb. 25, 2025. [Online]. Available: https://www.ncbi.nlm.nih.gov/books/NBK559063/ [3] B. Bakir, A. M. Chiarella, J. R. Pitarresi, and A. K. Rustgi, “EMT, MET, Plasticity, and Tumor Metastasis,” Trends Cell Biol, vol. 30, no. 10, pp. 764–776, Oct. 2020, doi: 10.1016/j.tcb.2020.07.003. [4] N. M. Aiello and Y. Kang, “Context-dependent EMT programs in cancer metastasis,” Journal of Experimental Medicine, vol. 216, no. 5, pp. 1016–1026, May 2019, doi: 10.1084/jem.20181827. [5] J. P. Thiery, G. Sheng, X. Shu, and R. Runyan, “How studies in developmental epithelial-mesenchymal transition and mesenchymal-epithelial transition inspired new research paradigms in biomedicine,” Development (Cambridge), vol. 151, no. 3, 2024, doi: 10.1242/dev.200128 [6] N. M. Aiello et al., “EMT Subtype Influences Epithelial Plasticity and Mode of Cell Migration,” Dev Cell, vol. 45, no. 6, 2018, doi: 10.1016/j.devcel.2018.05.027. [7] A. A. Bhat et al., “Tight Junction Proteins and Signaling Pathways in Cancer and Inflammation: A Functional Crosstalk,” Front Physiol, vol. 9, no. JAN, Jan. 2019, doi: 10.3389/fphys.2018.01942. [8] J. Jayachandran, H. Srinivasan, and K. P. Mani, “Molecular mechanism involved in epithelial to mesenchymal transition,” Arch Biochem Biophys, vol. 710, p. 108984, Oct. 2021, doi: 10.1016/j.abb.2021.108984. [9] S. Usman et al., “Vimentin Is at the Heart of Epithelial Mesenchymal Transition (EMT) Mediated Metastasis,” Cancers (Basel), vol. 13, no. 19, p. 4985, Oct. 2021, doi: 10.3390/cancers13194985. [10] Jonathan W Rick 2019, “Fibronectin in Malignancy: Cancer-Specific Alterations, Pro-Tumoral Effects, and Therapeutic Implications,” HHS Public Access, pp. 1–18, doi: DOI:10.1053/j.seminoncol.2019.08.002. [11] E. Sabouni et al., “Unraveling the function of epithelial-mesenchymal transition (EMT) in colorectal cancer: Metastasis, therapy response, and revisiting molecular pathways,” Biomedicine & Pharmacotherapy, vol. 160, p. 114395, Apr. 2023, doi: 10.1016/j.biopha.2023.114395. [12] M. Saitoh, “Involvement of partial EMT in cancer progression,” The Journal of Biochemistry, vol. 164, no. 4, 2018, doi: 10.1093/jb/mvy047. [13] D. Ribatti, R. Tamma, and T. Annese, “Epithelial-Mesenchymal Transition in Cancer: A Historical Overview,” Transl Oncol, vol. 13, no. 6, p. 100773, Jun. 2020, doi: 10.1016/j.tranon.2020.100773. [14] J. Yang et al., “Guidelines and definitions for research on epithelial–mesenchymal transition,” Nat Rev Mol Cell Biol, vol. 21, no. 6, pp. 341–352, Jun. 2020 [15] M. Rembold et al., “A conserved role for Snail as a potentiator of active transcription,” Genes Dev, vol. 28, no. 2, 2014, doi: 10.1101/gad.230953.113. [16] I. Gray, P. Szymanski, and M. Levine, “Short-range repression permits multiple enhancers to function autonomously within a complex promoter,” Genes Dev, vol. 8, no. 15, 1994, doi: 10.1101/gad.8.15.1829. [17] H. Peinado, D. Olmeda, and A. Cano, “Snail, ZEB and bHLH factors in tumour progression: An alliance against the epithelial phenotype?,” 2007. doi: 10.1038/nrc2131. [18] A. A. Postigo and D. C. Dean, “ZEB represses transcription through interaction with the corepressor CtBP,” Proc Natl Acad Sci U S A, vol. 96, no. 12, 1999, doi: 10.1073/pnas.96.12.6683 [19] S. Spaderna et al., “The transcriptional repressor ZEB1 promotes metastasis and loss of cell polarity in cancer,” Cancer Res, vol. 68, no. 2, 2008, doi: 10.1158/0008-5472.CAN-07-5682. [20] B. Thisse, M. El Messal, and F. Perrin-Schmitt, “The twist gene: Isolation of a Drosophila zygotle gene necessary for the establishment of dorsoventral pattern,” Nucleic Acids Res, vol. 15, no. 8, 1987, doi: 10.1093/nar/15.8.3439. [21] V. El Ghouzzi et al., “Mutations of the TWIST gene in the Saethre-Chotzen syndrome,” 1997. doi: 10.1038/ng0197-42. [22] H. L. Franco, J. Casasnovas, J. R. Rodríguez-Medina, and C. L. Cadilla, “Redundant or separate entities? - Roles of Twist1 and Twist2 as molecular switches during gene transcription,” Nucleic Acids Res, vol. 39, no. 4, 2011, doi: 10.1093/nar/gkq890. [23] C. W. Li et al., “Epithelial-mesenchymal transition induced by TNF-? requires NF-?B-mediated transcriptional upregulation of Twist1,” Cancer Res, vol. 72, no. 5, 2012, doi: 10.1158/0008-5472.CAN-11-3123. [24] F. Kroepil et al., “Down-Regulation of CDH1 Is Associated with Expression of SNAI1 in Colorectal Adenomas,” PLoS One, vol. 7, no. 9, 2012, doi: 10.1371/journal.pone.0046665. [25] C. P?a et al., “SNAI1 expression in colon cancer related with CDH1 and VDR downregulation in normal adjacent tissue,” Oncogene, vol. 28, no. 49, 2009, doi: 10.1038/onc.2009.285 [26] W. K. K. Wu et al., “Dysregulation and crosstalk of cellular signaling pathways in colon carcinogenesis,” 2013. doi: 10.1016/j.critrevonc.2012.11.009. [27] M. Bezdekova, S. Brychtova, E. Sedlakova, K. Langova, T. Brychta, and K. Belej, “Analysis of snail-1, E-cadherin and claudin-1 expression in colorectal adenomas and carcinomas,” Int J Mol Sci, vol. 13, no. 2, 2012, doi: 10.3390/ijms13021632. [28] A. Dongre and R. A. Weinberg, “New insights into the mechanisms of epithelial–mesenchymal transition and implications for cancer,” Nat Rev Mol Cell Biol, vol. 20, no. 2, pp. 69–84, Feb. 2019, doi: 10.1038/s41580-018-0080-4 [29] E. Batlle and J. Massagué, “Transforming Growth Factor-? Signaling in Immunity and Cancer,” 2019. doi: 10.1016/j.immuni.2019.03.024. [30] G. Chalkiadaki et al., “Fibroblast growth factor-2 modulates melanoma adhesion and migration through a syndecan-4-dependent mechanism,” International Journal of Biochemistry and Cell Biology, vol. 41, no. 6, 2009, doi: 10.1016/j.biocel.2008.11.008. [31] S. H. Shirley, V. R. Greene, L. M. Duncan, C. A. Torres Cabala, E. A. Grimm, and D. F. Kusewitt, “Slug expression during melanoma progression,” American Journal of Pathology, vol. 180, no. 6, 2012, doi: 10.1016/j.ajpath.2012.02.014 [32] H. Merikallio et al., “Snail promotes an invasive phenotype in lung carcinoma,” Respir Res, vol. 13, 2012, doi: 10.1186/1465-9921-13-104. [33] Yastrebova, I. Khamidullina, V. V. Tatarskiy, and Scherbakov, “Snail-Family Proteins: Role in Carcinogenesis and Prospects for Antitumor Therapy,” Acta Naturae, vol. 13, no. 1, 2021, doi: 10.32607/actanaturae.11062. [34] S. Mikami et al., “Expression of snail and slug in renal cell carcinoma: E-cadherin repressor snail is associated with cancer invasion and prognosis,” Laboratory Investigation, vol. 91, no. 10, 2011, doi: 10.1038/labinvest.2011.111. [35] M. Zivotic et al., “SLUG and SNAIL as Potential Immunohistochemical Biomarkers for Renal Cancer Staging and Survival,” Int J Mol Sci, vol. 24, no. 15, 2023, doi: 10.3390/ijms241512245. [36] E. Batlle et al., “The transcription factor Snail is a repressor of E-cadherin gene expression in epithelial tumour cells,” Nat Cell Biol, vol. 2, no. 2, 2000, doi: 10.1038/35000034. [37] A. Cano et al., “The transcription factor Snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression,” Nat Cell Biol, vol. 2, no. 2, 2000, doi: 10.1038/35000025. [38] B. S. Grimes, T. C. Walser, R. Li, Z. Jing, L. Tran, and S. M. Dubinett, “The transcription factor Slug induces diverse malignant phenotypes in models of established lung cancer and pulmonary premalignancy,” Journal of Thoracic Oncology, vol. 11, no. 2, 2016, doi: 10.1016/j.jtho.2015.12.036. [39] K. Shien et al., “Acquired resistance to EGFR inhibitors is associated with a manifestation of stem cell-like properties in cancer cells,” Cancer Res, vol. 73, no. 10, 2013, doi: 10.1158/0008-5472.CAN-12-4136 [40] E. R. Camp, J. E. Walsh, S. G. Vaena, D. N. Lewin, D. J. Cole, and D. K. Watson, “Slug Expression Enhances Tumor Formation In A Non-invasive Pre-clinical In Vivo Rectal Cancer Model,” Journal of Surgical Research, vol. 165, no. 2, 2011, doi: 10.1016/j.jss.2010.11.198. [41] S. Tanaka, W. Kobayashi, M. Haraguchi, K. Ishihata, N. Nakamura, and M. Ozawa, “Snail1 expression in human colon cancer DLD-1 cells confers invasive properties without N-cadherin expression,” Biochem Biophys Rep, vol. 8, 2016, doi: 10.1016/j.bbrep.2016.08.017. [42] N. Fenouille et al., “The epithelial-mesenchymal transition (EMT) regulatory factor SLUG (SNAI2) is a downstream target of SPARC and AKT in promoting melanoma cell invasion,” PLoS One, vol. 7, no. 7, 2012, doi: 10.1371/journal.pone.0040378. [43] H. H. Lee et al., “Evaluation of Slug expression is useful for predicting lymph node metastasis and survival in patients with gastric cancer,” BMC Cancer, vol. 17, no. 1, 2017, doi: 10.1186/s12885-017-3668-8. [44] D. J. Zhu et al., “Twist1 is a potential prognostic marker for colorectal cancer and associated with chemoresistance,” Am J Cancer Res, vol. 5, no. 6, 2015. [45] M. Khot et al., “Twist1 induces chromosomal instability (CIN) in colorectal cancer cells,” Hum Mol Genet, vol. 29, no. 10, 2020, doi: 10.1093/hmg/ddaa076. [46] M. Y. Feng et al., “Metastasis-induction and apoptosis-protection by TWIST in gastric cancer cells,” Clin Exp Metastasis, vol. 26, no. 8, 2009, doi: 10.1007/s10585-009-9291-6. [47] H. jie Zhang et al., “Twist2 promotes kidney cancer cell proliferation and invasion via regulating ITGA6 and CD44 expression in the ECM-Receptor-Interaction pathway,” Biomedicine and Pharmacotherapy, vol. 81, 2016, doi: 10.1016/j.biopha.2016.02.042. [48] H. Nakashima et al., “Involvement of the transcription factor twist in phenotype alteration through epithelial-mesenchymal transition in lung cancer cells,” Mol Carcinog, vol. 51, no. 5, 2012, doi: 10.1002/mc.20802 [49] F. Wang et al., “ZEB1 promotes colorectal cancer cell invasion and disease progression by enhanced LOXL2 transcription.,” Int J Clin Exp Pathol, vol. 14, no. 1, 2021. [50] M. Wang et al., “Potential role of ZEB1 as a DNA repair regulator in colorectal cancer cells revealed by cancer-Associated promoter profiling,” Oncol Rep, vol. 38, no. 4, 2017, doi: 10.3892/or.2017.5888. [51] J. E. Larsen et al., “ZEB1 drives epithelial-to-mesenchymal transition in lung cancer,” Journal of Clinical Investigation, vol. 126, no. 9, 2016, doi: 10.1172/JCI76725. [52] T. Zhang et al., “A genetic cell context-dependent role for ZEB1 in lung cancer,” Nat Commun, vol. 7, 2016, doi: 10.1038/ncomms12231. [53] G. J. Zhang, T. Zhou, H. P. Tian, Z. L. Liu, and S. Sen Xia, “High expression of ZEB1 correlates with liver metastasis and poor prognosis in colorectal cancer,” Oncol Lett, vol. 5, no. 2, 2013, doi: 10.3892/ol.2012.1026. [54] M. Li, H. Guan, Y. Liu, and X. Gan, “LncRNA ZEB1 AS1 reduces liver cancer cell proliferation by targeting miR 365a 3p,” Exp Ther Med, 2019, doi: 10.3892/etm.2019.7358. [55] H. Shen et al., “ZEB1-induced LINC01559 expedites cell proliferation, migration and EMT process in gastric cancer through recruiting IGF2BP2 to stabilize ZEB1 expression,” Cell Death Dis, vol. 12, no. 4, 2021, doi: 10.1038/s41419-021-03571-5. [56] Q. C. C. T. Sheng Lin, “ZEB family is a prognostic biomarker and correlates with anoikis and immune infiltration in kidney renal clear cell carcinoma,” BMC Med Genomics, 2024 [57] C. Lindskog, “The Human Protein Atlas – an important resource for basic and clinical research,” 2016. doi: 10.1080/14789450.2016.1199280 [58] P. J. Thul and C. Lindskog, “The human protein atlas: A spatial map of the human proteome,” Protein Science, vol. 27, no. 1, 2018, doi: 10.1002/pro.3307. [59] A. Pavli?, N. Hauptman, E. Boštjan?i?, and N. Zidar, “Long Non-Coding RNAs as Potential Regulators of EMT-Related Transcription Factors in Colorectal Cancer—A Systematic Review and Bioinformatics Analysis,” 2022. doi: 10.3390/cancers14092280. [60] A. Digre and C. Lindskog, “The human protein atlas—Integrated omics for single cell mapping of the human proteome,” Protein Science, vol. 32, no. 2, 2023, doi: 10.1002/pro.4562.

Copyright

Copyright © 2025 Supriya Soni, Rose Rani, Archana Tiwari, Dipanjana Ghosh. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.