Sex Steroid Receptors in Colorectal Cancer: Implications for Tumor Progression, Therapeutic Opportunities, and Sex-Specific Outcomes

Sex Steroid Receptors in Colorectal Cancer: Implications for Tumor Progression, Therapeutic Opportunities, and Sex-Specific Outcomes | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 2 January 2025 V1 Latest version Share on Sex Steroid Receptors in Colorectal Cancer: Implications for Tumor Progression, Therapeutic Opportunities, and Sex-Specific Outcomes Authors : Shreya Singh Beniwal , Srajan Gupta , Indu Etta , Yash Janu , Sidharth Olikkal S 0009-0004-6062-8139 , Kareema Cummings , Roma Patil , Saif Syed , Vaishnavi Rajesh Shetty , and Ayush Dwivedi 0009-0005-1955-9116 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173580151.15154655/v1 Published Annals of Medicine & Surgery Version of record Peer review timeline 1068 views 165 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background : Colorectal cancer is a common malignancy characterised by the abnormal growth of cells in the colon or rectum. Its high occurrence and death rate make it a significant concern for public health. Hormone signalling pathways including estrogen, progestrone and androgen receptors have gained attention in CRC research for their potential impact on tumor development and progression and open possibilities for newer therapeutic approaches. Objective : The objectives of the study are to investigate the role of sex steroid receptors in colorectal cancer, to explore the potential therapeutic opportunities in CRC treatment, and to discuss the implications of sex specificity in study design and interpretation. Method : Multiple cohort studies were dissected to provide valuable insights into the intricate world of sex steroid receptors in CRC. The results of the cohort studies were analysed based on gender, age, clinical stage and anatomical location. Results : The study emphasizes the intricate roles of sex hormones and their receptors in colorectal cancer (CRC) progression, highlighting that ERβ has anti-tumor effects with lower levels linked to colonic tumors in females, while ERα and androgen receptors (AR) promote growth, particularly in postmenopausal women. Progesterone receptors are linked to poorer prognosis, though progesterone treatment inhibits CRC cell proliferation. In malignant tissues, ERα and AR levels increase, while ERβ and progesterone receptors decrease. ER isoforms’ mRNA levels are lower in malignant female cases, while AR expression is higher in males. Additionally, the location of CRC differs by sex, with women more likely to develop proximal colon cancer—associated with reduced ERβ—while men tend to develop distal CRC. This research reveals anuanced modulation of gene transcription in CRC by ERα, ERβ, and GPER, reflecting both tumor-promoting and suppressive effects of these hormones. Limitations : The study highlights that the mechanism by which progesterone reduces colorectal cancer (CRC) progression remains uncertain. Limitations include variability in findings due to differences in cell lines, hormone concentrations, and receptor expression. The study also notes inconsistencies across research on hormone replacement therapy (HRT) and its potential to reduce CRC risk, suggesting a need for standardized methodologies to evaluate the progesterone receptor’s impact on CRC prognosis. Additionally, it acknowledges the complex interplay between cytokines in the tumor microenvironment and estrogen signaling, creating a challenging feedback loop. Future directions: The article examines emerging therapeutic strategies in colorectal cancer (CRC), suggesting that combined activation of Estrogen Receptor Beta (ERβ) and Progesterone Receptor (PGR) could produce anti-cancer effects. It proposes sequential estrogen-progesterone therapy as a promising regimen for early-stage CRC, while simultaneous therapy may benefit advanced cases. Future research should clarify the role of sex hormones in CRC development, advance prognostic markers, and explore selective estrogen receptor modulators (SERMs) as potential therapies. It also calls for investigation into pharmacological agents targeting ERβ and the influence of the gut microbiome in CRC prevention, paving the way for tailored therapeutic interventions. Sex Steroid Receptors in Colorectal Cancer: Implications for Tumor Progression, Therapeutic Opportunities, and Sex-Specific Outcomes Shreya Singh Beniwal 1* , Srajan Gupta 2* , Indu Etta 3* , Yash Janu 4* , Sidharth S Olikkal 5* , Kareema Cummings 6 * , Roma Patil 7* , Saif Syed 8* , Vaishnavi Rajesh Shetty 9* , Ayush Dwivedi 10* . 1. Lady Hardinge Medical College, Connaught Place, New Delhi, India - 110001; [email protected] 2. Sri Venkateswara Medical College, Alipiri Road, Tirupati, Andhra Pradesh, India, Pin - 517507; [email protected] 3. Kakatiya Medical College, Rangampeta Street, Nizampura, Warangal, Telangana, Pin - 506007; [email protected] 4. Government Medical College, Kota, Rangbari, RK Puram, Kota; [email protected] 5. Government Medical College, Thiruvananthapuram, Medical College Junction, Ulloor, Thiruvananthapuram, Pin - 695011; [email protected] 6. Millennium Heights Medical Complex, Castries, Saint Lucia; [email protected] 7. Bangalore Medical College and Research Institute, 85, Byrathi, Hennur Main Road, Bangalore, Pin - 560077; [email protected] 8. Royal College of Surgeons Ireland, Dublin, Ireland - Pin 214; [email protected] 9. Danylo Halytsky Lviv National Medical University, Lviv, Ukraine, Pin - 79000; [email protected] 10. Danylo Halytsky Lviv National Medical University, Lviv, Ukraine, Pin - 79000; [email protected] Abstract Background : Colorectal cancer is a common malignancy characterised by the abnormal growth of cells in the colon or rectum. Its high occurrence and death rate make it a significant concern for public health. Hormone signalling pathways including estrogen, progestrone and androgen receptors have gained attention in CRC research for their potential impact on tumor development and progression and open possibilities for newer therapeutic approaches. Objective : The objectives of the study are to investigate the role of sex steroid receptors in colorectal cancer, to explore the potential therapeutic opportunities in CRC treatment, and to discuss the implications of sex specificity in study design and interpretation. Method : Multiple cohort studies were dissected to provide valuable insights into the intricate world of sex steroid receptors in CRC. The results of the cohort studies were analysed based on gender, age, clinical stage and anatomical location. Results : The study emphasizes the intricate roles of sex hormones and their receptors in colorectal cancer (CRC) progression, highlighting that ERβ has anti-tumor effects with lower levels linked to colonic tumors in females, while ERα and androgen receptors (AR) promote growth, particularly in postmenopausal women. Progesterone receptors are linked to poorer prognosis, though progesterone treatment inhibits CRC cell proliferation. In malignant tissues, ERα and AR levels increase, while ERβ and progesterone receptors decrease. ER isoforms’ mRNA levels are lower in malignant female cases, while AR expression is higher in males. Additionally, the location of CRC differs by sex, with women more likely to develop proximal colon cancer—associated with reduced ERβ—while men tend to develop distal CRC. This research reveals anuanced modulation of gene transcription in CRC by ERα, ERβ, and GPER, reflecting both tumor-promoting and suppressive effects of these hormones. Limitations : The study highlights that the mechanism by which progesterone reduces colorectal cancer (CRC) progression remains uncertain. Limitations include variability in findings due to differences in cell lines, hormone concentrations, and receptor expression. The study also notes inconsistencies across research on hormone replacement therapy (HRT) and its potential to reduce CRC risk, suggesting a need for standardized methodologies to evaluate the progesterone receptor’s impact on CRC prognosis. Additionally, it acknowledges the complex interplay between cytokines in the tumor microenvironment and estrogen signaling, creating a challenging feedback loop. Future directions: The article examines emerging therapeutic strategies in colorectal cancer (CRC), suggesting that combined activation of Estrogen Receptor Beta (ERβ) and Progesterone Receptor (PGR) could produce anti-cancer effects. It proposes sequential estrogen-progesterone therapy as a promising regimen for early-stage CRC, while simultaneous therapy may benefit advanced cases. Future research should clarify the role of sex hormones in CRC development, advance prognostic markers, and explore selective estrogen receptor modulators (SERMs) as potential therapies. It also calls for investigation into pharmacological agents targeting ERβ and the influence of the gut microbiome in CRC prevention, paving the way for tailored therapeutic interventions. Keywords: colorectal cancer (CRC), sex steroid receptors, estrogen receptor beta (ERbeta), progesterone receptor (PGR), androgen receptor (AR), tumor progression, estrogen-progesterone therapy Introduction Colorectal cancer (CRC) presents a major challenge in oncology, ranking globally as the second leading cause of cancer-related deaths and the third most prevalent cancer. Approximately 1.93 million new CRC cases were diagnosed in 2020, resulting in around 0.94 million deaths. With an estimated 3.2 million new cases expected by 2040, these projections indicate a substantial increase [1]. A highly processed Western diet, tobacco use, and obesity continue to play a pivotal role in colorectal cancer development [2]. In contrast, non-modifiable risk factors, like gender, have also been associated with varying susceptibility to colorectal polyps and tumors, with males exhibiting a higher risk for both [3] . This gender disparity has prompted the hypothesis that sex hormones may contribute to observed differences in colorectal cancer risk, especially when comparing pre-menopausal and post-menopausal women [4, 5] . Consistent epidemiological findings have indicated that pre-menopausal women have a lower risk of CRC development. Compared to nonusers and age-matched men, post-menopausal women using hormone replacement therapy (HRT) also exhibit a significantly reduced incidence [2, 6, 7] . Additionally, female CRC patients aged 18-44 years tend to have a more favorable prognosis compared to women over 50 years and men of the same age [8]. While research suggests that the primary production of sex steroid hormones occurs in the gonads, various peripheral tissues, including the colon, not only express enzymes necessary for sex hormone biogenesis, such as progesterone (P4), testosterone, and 17β-estradiol (E2) [9–11] , but also respond to sex hormones, as evidenced by the presence of estrogen receptors (ERα & ERβ) [12–14] , progesterone receptor (PGR) [15–17] , and androgen receptor (AR) [18–20] . Overall, the authors suggest that E2, acting through ERβ [21–24] , and P4 via PGR [24, 25] , may function as tumor suppressors. Meanwhile, the activation of ERα by E2 [21–24] and AR by testosterone [26–29] could potentially promote the development and progression of colon neoplasia [30]. This study aims to explore and compile existing research on the effects of estrogen, progesterone, and androgens on CRC, the molecular mechanisms of sex hormone-mediated chemoprotection, and the clinical implications of sex hormones in CRC. Better prognostic markers and/or alternative hormonal therapies for CRC can be provided if we understand the role of sex steroid hormones in colon oncogenesis. Role of Estrogen Receptor in Colorectal Carcinoma Colorectal cancer shows variable expression in men and women, while post-menopausal women do not exhibit this variability. Post-menopausal women taking hormone replacement therapy show a lower risk for colorectal cancer. This variable incidence of colorectal cancer is attributed to estrogen and the variable expression of estrogen receptors in the colonic mucosa. Estrogens take three different forms: Estrone (E1), Estradiol (E2), and Estriol (E3), whose roles are mostly mediated by two nuclear receptors (ERα and ERβ) and a membrane-associated G- protein (GPR30 or GPER) [32] . Estrogen response through signaling by ERα and ERβ depends on the ERα/ERβ ratio in a cell, as ERβ inhibits the activity of ERα [32] . While Erα predominantly influences reproductive tissues, ERβ has a broader tissue distribution, extending its influence beyond reproduction. ERβ mainly functions in maintaining epithelial structure, regulating gastrointestinal physiology, and mediating immunological responses [33, 34] . ERα promotes cellular growth and proliferation, while ERβ inhibits cellular proliferation by down-regulating proto-oncogenes and up- regulating tumor suppressor genes [35] . The distribution and expression of these receptors vary in normal and cancerous colon tissues. According to Xie et al., the expression of ERβ is higher in normal colonic tissue (91.7%) than in CRC tissue (83.3%), while the expression of ERα is higher in CRC tissue (25%) than in normal colonic mucosa (16.6%) [36, 37] . These contradicting effects of ERα and Erβ are evidenced by the use of selective estrogen receptor modulators (SERMs); ERα agonists increase the risk of colon cancer, while ERβ agonists decrease the risk [38, 39] . Expression of ERβ decreases as the stage of cancer increases [32] . Figure 1: Estrogen receptor stimulation affects gene transcription via interaction with specific gene sequences called estrogen response elements (EREs) and other transcription factors like c-Jun and c-Fos of activating protein-1 complex (AP1), stimulating protein 1 (Sp1), and nuclear factor-κB (NFκB) Estrogen receptor stimulation affects gene transcription via interaction with specific gene sequences called estrogen response elements (EREs) and other transcription factors like c-Jun and c-Fos of the activating protein-1 complex (AP1), stimulating protein 1 (Sp1), and nuclear factor-κB (NFκB) [32,40] . Transcription activation of a variety of genes, such as VEGF (angiogenesis), cadherins, laminins (cell adhesion), and anti-apoptotic genes, leads to tumor progression [32] . Estrogen binding to ERα activates gene transcription, while ERβ inhibits gene transcription [32] . Thus, the outcome effect of estrogen depends on the ratio of ERα and ERβ in colonic tissue. Genomic effects can occur even in the absence of estrogen ligands, where ER is stimulated via phosphorylation through the activated kinase pathway [32] . ERα has a monoubiquitination site that mediates the PI3K/Akt pathway; mutations in this site prevent phosphorylation of ERα [38, 41]. MAPK and PI3K/Akt signaling pathways play significant roles in tumorigenesis by increasing cellular growth and invasion [38, 42] . The ESR2-CA microsatellite in the estrogen receptor (ER)-β gene is associated with colorectal cancer risk. A shorter allele increases risk in older women but decreases it in younger postmenopausal women. In postmenopausal women, ESR2-CA genotypes and ER-β expression impact cancer characteristics based on age, location, and mismatch repair protein status. Noncancerous tissue from older women with specific genotypes exhibits higher ER-β expression. These findings suggest that germline ESR2-CA genotypes influence ER-β expression and may contribute to the clinical characteristics of colon cancer [36] . Chronic inflammation of the colon increases colorectal cancer (CRC) risk, and hormone-replacement therapy with estrogen receptor beta (ERβ) has protective effects. A study by Ibrahim A et al. investigated how intestinal Erβ influences the gut microbiota using mouse models of colitis-induced CRC. Loss of ERβ intensified the reduction in microbiota diversity caused by colitis-induced CRC. The Prevotellaceae_UCG_001 genus of Bacteroidetes was overrepresented in CRC mice, particularly in females and those lacking ERβ [43]. G-protein coupled estrogen receptor (GPER) is a transmembrane receptor. GPER exerts many cellular functions like proliferation, apoptosis, endoplasmic reticulum stress, angiogenesis, and immune response [44] . Expression of GPER is markedly less in cancerous tissue compared to adjacent normal tissue. GPER activation leads to decreased proliferation, increased endoplasmic reticulum stress, G2/M phase arrest, and promotes apoptosis in cancerous cells [45] . Estrogen regulates the expression of the ATM gene through GPER, which is more pronounced in hypoxic conditions. This establishes the relationship between estrogen and oxygen levels [38] . According to Jacenik D et al., GPER stimulation inhibits CRC cell migration in normoxic conditions and promotes migration in hypoxic conditions. GPER stimulation plays a role in colonic motility, suggesting that decreased stimulation could cause constipation [46] . Roberts MC et al. found a direct relationship between the risk of both benign and malignant colon cancer and the severity of constipation [47] . ERβ is proven to have anti-tumorigenic effects by regulating the cell cycle, increasing DNA repair capacity, and downregulating oncogenes like MYC and PROX1 [46]. Increased expression of ERβ in colonic tissues correlates with increased p53 signaling, which enhances apoptosis and decreases cell proliferation [46] . The concentration of ERβ in the colon could serve as a prognostic factor for CRC. ERβ levels are denser in normal colonic mucosa and significantly reduced with the progression of tumor staging [38, 39] . Figure 2: ERβ(estrogen receptor beta) acts as an important regulator of cell proliferation and immune function, generally promoting anti-inflammatory and immunosuppressive effects. Its activation or upregulation may have therapeutic potential in inflammatory and autoimmune conditions. [43][62][63] Overall, ERβ contributes to a more favorable microbiome that could mitigate CRC development by influencing metabolic functions and immune responses. Thus, this beneficial effect of estrogen on colonic tissue via estrogen receptors represents a potential therapeutic option for reducing the morbidity associated with colorectal cancer. Phytoestrogens are plant-derived estrogens that have been shown to have beneficial effects on colorectal cancer. Role of Progesterone Receptor in Colorectal Carcinoma. The progesterone receptor’s involvement in colorectal cancer (CRC) remains an area of exploration, with emerging evidence pointing to its potential influence on cell differentiation and tumor progression. The role of progesterone receptor expression in colorectal neoplasms is contradictory. The progesterone receptors in colorectal carcinoma are not very frequent but are much more frequent than estrogen receptors; however, the receptor content is generally low [48] . Some authors have found that the amount of progesterone receptors differs in the colon and rectum, with higher content in the colon tumor cells [48,49] . This can in no way be compared to the receptors and content in breast carcinoma, which is much higher, even though both tumors suggest similar etiological factors related to a high-fat and protein diet [50] . In a study conducted by Zhang et al., low levels of progesterone receptors were associated with a poor prognosis in colorectal cancer. Treatment with increased concentrations of progesterone showed that there was an inhibition of CRC cell proliferation in vitro and in vivo, as well as an upregulation of the JNK pathway via GADD45α to reduce the malignant progression of colorectal carcinoma [51] . The apoptosis of colorectal cancer cells is related to the upregulation of GADD45α and the activation of mitogen-activated protein kinase (JNK) [56] . However, the exact mechanism by which progesterone reduces the malignant progression of colorectal carcinoma remains unclear. Progesterone can attach to nuclear or membrane receptors via classical or non-classical pathways, thereby regulating tumor growth. In a non-classical pathway, the progesterone response elements bind to proto-oncogene tyrosine-protein Src, protein kinase B, and mitogen-activated protein kinase, with subsequent actions on effector targets like wingless-type MMTV integration site family member 1, cyclin D1, epidermal growth factor receptor, and transcription of p21. The classical pathway leads to the decomposition of heat shock protein, progesterone receptor dimerization, and progesterone response elements initiating effector targets like cyclin D1 and p4 mediator receptor activator of nuclear factor κB ligand [57] . Another study suggests an inverse association between cancer recurrence and the PR pathway. The PR pathway was significantly associated with advanced cancer stages and response to adjuvant chemotherapy [52] . In contrast, some studies have found no evidence of a correlation between progesterone receptors and the prognosis of colorectal cancer [53, 54] . The variations obtained could be due to several factors, including the methods used for sample collection, the method of staining, the level of staining considered positive, and the location of the sample [55] . Role of Androgen Receptors in Colon Carcinoma Androgen exhibits its effect on colonic tissue through nuclear receptors and membrane-bound androgen receptors. Androgen receptors (AR) are nuclear receptors that alter gene expression by binding to DNA [58] . AR expression varies in normal and cancerous colonic tissue. There is almost no expression of AR in normal colonic tissue, while its expression increases as normal cells differentiate into cancerous cells [58] . A significant correlation exists between AR expression and tumor characteristics (size, cell differentiation, and AJCC staging) [58] . According to Albasri AM et al., increased AR expression correlates with increased tumor size (size greater than 4 cm showed higher AR expression than size below 4 cm [pvalue=0.026]), poor cell differentiation, advanced staging, lymph node positivity, and distant metastasis [31, 58] . AR expression acts as a prognostic indicator, with increased expression shown to have decreased survival [58] . Gender and age showed variations in AR expression. According to Refaat B et al., AR expression is higher in right-sided early- stage cancer in men than in women, while the expression in the left-sided colon is almost equal in both men and women [31] . Women aged less than 50 years showed a lower expression of AR than women older than 60 [31] . Membrane androgen receptors (mAR) are membrane-bound receptors that affect colonic tissue by non-genomic signaling [59] . mAR activation shows tumor-suppressing effects, which is in contrast to the nuclear androgen receptors [59] . Non-permeable testosterone-albumin conjugates (TAC) bind to mAR,leading to rapid cytoskeleton reorganization (actin and tubulin) and promoting apoptosis through pro-apoptotic executor caspase-3 activation [59] . Anti-androgens cannot block the effects of mAR, as the pathways through which they act are different from those of nuclear androgen receptors [60] . mAR expression is increased in cancerous colonic tissue, while it is undetectable in normal colonic tissue [60] . Activation of mAR through TAC has antitumorigenic effects by inducing late downregulation of pro-survival PI-3K/Akt pathways, downregulation of c-Src phosphorylation (upregulation of c-Src activity shows tumor progression in the colonic tissue and metastasis), late GSK-3beta phosphorylation (increased apoptotic signaling), and late downregulation of the beta-catenin gene [61] . The pro-apoptotic responses of membrane androgen receptors are invalidated by the overexpression of anti-apoptotic factors by cancer cells [59] . Cross-talk and Interplay of Receptors ER and colorectal cancer Cross-talk between signaling cascades is a complicated interaction between estrogen receptors (ERs) and other signaling pathways that is critical in the colorectal cancer (CRC) process and therapy. Both genomic and non-genomic processes are involved in estrogen signaling pathways. Estrogen binds to ERs, where it interacts with DNA and modifies gene expression, among other genomic functions. On the other hand, non- genomic effects affect processes like cell migration and signaling, causing quick biological reactions. By forcing ERs to interact with DNA, estrogen binding modifies gene expression and protein synthesis in the genomic route. Membrane-bound ERs in the non-genomic pathway initiate a swift intracellular signaling cascade that affects cell functions such as motility and signaling [62, 63] . Besides genomic and non-genomic pathways, there is building awareness of convergent pathways that include both components. Two mechanisms for ”cross-talk” have been found. In one, estrogen-bound nuclear estrogen receptor complexes dimerize and translocate to the nucleus, where they interact with phosphorylated transcription factors activated by GPER1 signaling. Second, interactions between GPER1 and ERα/ERβ at the plasma membrane activate protein kinase cascades, resulting in the phosphorylation of transcription factors, including estrogen receptors. These phosphorylated factors can theninteract with DNA sequences, regulating transcription [64] . The exploration of these intricate signaling pathways holds substantial pharmacological potential, as their modulation could pave the way for innovative treatments for colorectal cancer. According to Francesco Caiazza et al., E2 promotes Erbeta mRNA translation in the near term (2 and 4 hours after stimulation) followed by late increased transcription (24 hours after stimulation). E2-induced sustained and palmitoylation- dependent p38/MAPK activation was necessary for both processes. The findings also point to a highly tuned control of diverse cellular molecular activities by fast signals, which is necessary for E2’s protective actions against colon cancer progression. The interplay between these subtypes affects processes like cell proliferation and survival, with implications for diseases such as breast cancer and even colorectal cancer [65] . A study by Edvardsson K et al. also suggests that estrogen’s protective role against colon cancer is primarily mediated by estrogen receptor β (ERβ). ERβ expression inhibits colorectal cancer growth in xenografts. Reintroducing ERβ in three cancer cell lines led to cell-specific gene regulation, impacting apoptosis, cell differentiation, and cell cycle regulation. Notably, ERβ down-regulated IL-6 and associated networks, impacting inflammation in colon cancer development. ERβ and the nuclear receptor co-regulator PROX1 share target genes, while ERβ also enhances DNA-repair capacity, indicating anti-tumorigenic effects. Enhancing ERβ action could offer a promising therapeutic avenue for colon cancer prevention and treatment [43] . AR and CRC Androgen receptors play a complex part in the regression of colorectal cancer (CRC), either independently or in conjunction with other sex hormones. According to Roshan et al., the activation of androgen receptors (AR) is notably more prevalent in CRC tissue when compared to normal colon tissue. Activation of AR by testosterone-HSA conjugates triggers apoptosis, offering a protective mechanism against CRC. The invasiveness of CRC is under the control of Akt kinases, which are activated by testosterone-HSA, thereby curbing tumour invasion. Additionally, the number of CAG repeats in the AR gene is linked to survival rates, with longer repeats associated with less favourable outcomes. The CARM1 protein, which is required for cell proliferation and survival, is overexpressed in CRC; however, its exact role is unknown [67] . Xia T et al. discovered a significant association between hypomethylation at specific sites (cg17964359 and cg18156601) in the AR gene and an increased risk of CRC. This hypomethylation in peripheral blood leukocytes (PBL) may even serve as a potential biomarker for CRC [68] . PR and CRC Studies have also shown that progesterone (P4) has strong anti-cancer properties in the setting of colon cancer. When used as a monotherapy, P4 has been shown to inhibit the proliferation of colon cancer cells in both in vitro and xenograft models. This anti-proliferative activity is connected with the arrest of the cell cycle at the G0/G1 phase, which reduces cell division and tumour formation. Furthermore, P4 has been shown to trigger apoptosis in colon cancer cells, which is a programmed cell death mechanism that effectively controls cancer cell proliferation. Moreover, P4 inhibits cell proliferation and promotes death in colon cancer cells by activating the JNK pathway and increasing the production of GADD45α, an antiproliferative and DNA-damage-inducible protein. The potential of P4 as a promising therapeutic agent in the treatment of colon cancer is highlighted by these collective findings [69] . Kamińska et al. found considerable down-regulation of progesterone receptor (PGR), membrane progesterone receptors (mPRβ and mPRγ), and PGRMC2 in CRC tissues compared to normal tissues. This decreased expression of PGR is related to a poorer prognosis for CRC. In contrast, the high expression of PGRMC1 in later stages of CRC suggests a potential role in cancer growth. Furthermore, their findings demonstrate that progesterone (P4) therapy can reduce the proliferation of numerous colorectal cancer cell lines by halting the cell cycle at the G2/M phase and triggering apoptosis. These findings highlight the intricate link between progesterone receptors and CRC, and the potential utility of P4 in limiting the proliferation and advancement of CRC cells [70] . Zhang YL et al. propose that progesterone’s capacity to impede the S and G2/M stages of the cell cycle, downregulate specific cell cycle-related proteins, and induce apoptosis underscores its therapeutic potential. Notably, the study emphasizes the variability in research outcomes, which can be attributed to differences in cell lines, progesterone concentrations, and progesterone receptor (PGR) expression. Progesterone seems to exert its effects through both classical and non-classical pathways, with classical pathways involving interactions with various signaling molecules like mitogen-activated protein kinases (MAPKs), and non-classical pathways involving interactions with cyclin D1 and other downstream effectors. The induction of apoptosis by progesterone is intricately linked to GADD45 and the activation of the MAPK pathway, further highlighting its potential to inhibit the growth of colorectal carcinoma (CRC) [51] . Furthermore, research suggests that the progesterone (PGR) and estrogen receptor beta (ERβ) pathways interact in CRC, with PGR’s anti-tumorigenic actions dependent on ERβ activity in malignant tissues [52]. Figure 3: Schematic overview of the potential non-genomic P4 action in colorectal cancer. P4 may initiate rapid non-classical signalling through the complex of PGRMC1 and NENF, leading to increased proliferation and invasion of colorectal cancer cells. However, P4 cannot activate the classical genomic signalling pathway due to weak PGR expression in colorectal cancer cells (arrow: ↓PGR—weak PGR expression). P4 or NENF may significantly increase the release of IL-8 by colorectal cancer cells (arrow: ↑IL-8—increased release of IL-8). P4 significantly up-regulates mPRα and mPRγ expression in colorectal cancer cells (arrow: ↑mPR-α, ↑mPR-γ—increased expression of mPR-α and mPR-γ). NENF, neuron-derived neurotrophic factor; P4, progesterone; PGR, nuclear progesterone receptor; and PGRMC1, progesterone receptor membrane component 1. Methodology and Design This section details the methodologies employed in two cohort studies conducted by Giralda Topi et al. and Refaat B et al. It describes the patient populations, sample collection methods, immunohistochemistry techniques, and study aims. G. Topi et al. used random selection to investigate female patients between January 2008 and June 2012 who underwent primary CRC surgery. The study included 333 female patients who were diagnosed with primary colorectal cancer between 2008 and 2012 [71] . The research comprised female patients with a primary diagnosis of CRC who were both physically and psychologically capable of participating. Tissue microarrays were performed on 320 original CRC tumour samples using a monoclonal anti-ERB antibody. An immunohistochemistry procedure was conducted to assess the intensity of staining. Their research aimed to determine the relationship between Erβ expression and overall survival, disease-free survival, hormone status, lifestyle, and their effects [71] . Comparatively, the study conducted by Refaat B et al. utilized archived paired normal and malignant colon specimens that were collected from 120 patients (Saudi males and females) between January 2019 and December 2021. The specimens were formalin-fixed paraffin-embedded. The study included patients who were either over 18 but under 50 years old or over 60 year old. All participants had been diagnosed with primary sporadic cancer and had not received neoadjuvant chemotherapy or radiotherapy before their surgery. Immunohistochemistry was employed to quantify the levels of ERα, ERβ, PGR, and AR proteins [31] . Immunohistochemistry was performed using primary mouse monoclonal IgG antibodies to identify ERα, ERβ, PGR, and AR in 5-µm slices of both benign and cancerous tissue. Endogenous peroxidases were inhibited by immersing the sample in a BLOXALL solution for 15 minutes. The sections were thereafter placed in an incubator and left overnight in the presence of the primary antibodies. Following the washing process, the sections were subjected to treatment with ImmPRESS HRP Horse Anti-Mouse IgG Plus Polymer Peroxidase Kiss, by the instructions provided by the manufacturer. The same technique was used for the non-malignant regions. The quantification of protein expression was conducted using the IHC Image Analysis Toolbox. Regions of interest (ROIs) were identified, and the intensity of staining, along with the proportion of stained areas, was quantified. Subsequently, immunohistochemistry (IHC) scores were calculated for each receptor. These IHC scores were compared between matched normal and malignant tissues from each patient. Further analyses included comparisons acros different clinical stages (early [I/II] vs. late [III/IV]), genders (male vs. female), tumour locations (right-sided colon [RSC] vs. left-sided colon [LSC]), and age groups (≤ 50 vs. ≥ 60 years). This analysis was conducted on a total of 31 patients, with findings categorized by gender, age, clinical stage, and anatomical site (right: RSC vs. left: LSC). The research also aimed to evaluate the impact of hormone proteins on the cellcycle and apoptosis in male and female colorectal cancer cell lines SW480 and HT29. Hormone levels were assessed both individually and in combination with their respective inhibitors: ERα (MPP dihydrochloride), ERβ (PHTPP), PGR (mifepristone), and AR (bicalutamide) [ 31] . G. Topi et al. were thorough in describing the study methodologies; however, they could have benefitted from providing more clarity on certain aspects such as patient selection criteria and immunohistochemistry protocols to ensure reproducibility and transparency in the research methods, as seen by Refaat B et al. Effects of the hormone receptor blockers A significant increase in the numbers of SW480 male and HT29 female colon cancer cell lines in the sub-G1 phase was found after single treatments of E2 and P4 hormones compared. untreated cells. There was a significant increase in percentage of cells relative to non-treated cells in the sub-G1 phase with ERa blocker [MPP]. In contrast, there was a remarkable decline of SW480 and HT29 cell lines in the sub-G1 phase compared with cells treated with E2 and P4 monotherapies alone with the addition of ERb blocker [PHTPP] and PGR blocker [Mifepristone] [31] . Cellular apoptosis There was an increase in the numbers of early and late apoptotic cells relative to untreated cells on single treatment with E2. Era blocker enhanced the pro-apoptotic effects of E2, whereas, ERb blocker inhibited the pro-apoptotic effects of E2 in both cell lines. The numbers of viable SW480 and HT29 cells were significantly enhanced with testosterone monotherapy, while the addition of bicalutamide showed increased apoptosis in both cell lines [31] . Gender-specific variations ERb and PGR IHC scores correlated indirectly with N stage, M stage, number of positive lymph nodes, and advanced cance stage in both males and females. PGR, but not ERb, correlated indirectly with older age and T stage in malignant female specimens [31] . It has been indicated in many studies that ERT (estrogen replacement therapy) reduces colon cancer risk in postmenopausal women. Comparative reverse transcription-PCR and then southern analysis were done in a study to detect the level of mRNA expression levels for ER subtypes in paired samples of colon tumours and normal mucosa. In female patients, ERbeta steady-state levels were significantly decreased in colon tumours compared with normal mucosa. Levels of both ERbeta1 and ERbeta2 isoforms were significantly decreased in tumours from female patients, with a more remarkable decline in ERbeta1 mRNA levels. ERa mRNA levels were much lower than ERb levels in both genders. In some colon cancer cell lines (Caco-2, T84, and SW1116), ERb mRNA was detected while all other cell lines were negative for Era mRNA. The study showed ERb as the predominant ER subtype in the human colon, which suggests that decreased levels could be associated with the development of colonic tumors in females [73] . In almost all samples, analysis of AR in neoplastic and surrounding healthy tissues showed specific binding for DHT, demonstrating the presence of AR. Hence, no significant difference was observed between males and females and between healthy and neoplastic tissues [74]. Using western blot, AR was further characterized, and both AR isoforms: AR-B and AR-A, were detected in healthy mucosa while only the AR-A isoform was detected in neoplastic mucosa [74] . Menopausal status Studies have shown that colon cancer risk in postmenopausal women is affected by ERb gene (ESR2) cytosine-adenine (CA) repeat polymorphism (ESR2-CA) in the germline [77]. A different pathogenic role of this polymorphism is seen with age, as a shorter allele of this polymorphism is associated with a higher risk in older women, but lower risk in younger postmenopausal women. ESR2-CA is a microsatellite region [78] . A close relation was found between right-sided tumours and the level of ERb positivity and E2 concentration in women above the age of 70. On the other hand, a reduction in ERb compared to non-cancerous counterparts was only observed in left-sided tumours in women below the age of 70 [65] . Studies have reported that the germline ESR2-CA repeat polymorphism of the ER-β gene has an effect only on colon cancer risk in postmenopausal women but not on rectal or colon cancer risk in either men or pre-menopausal women [65] . The expression of androgen receptors was significantly higher in RSC and LSC malignant specimens obtained from post-menopausal women relative to premenopausal women [65] . In general, ERa expression increased significantly in malignant vs non-malignant specimens. However, left-sided cancers have higher ERa expression than right-sided colon cancers. Concerning the clinical stage, markedly higher IHC scores were seen in the late-stage right and left cancers than in their corresponding early-stage cancerous tumours [31] . In non-cancerous tissues, PGR expression was significantly higher in the left-sided compared to right-sided tumours, whereas in cancerous tissues, PGR expression declined remarkably, with left-sided tumours showing the highest decline relative to the right-sided tissues. While androgen receptor expression increased significantly in cancerous colonic tissues as compared to non-cancerous tissues, its expression was equal between proximal and distal cancers [31] . Implications of sex steroid receptor profiles Estrogens have been implicated in different non-endocrine-related cancer types such as lung and gastrointestinal [71] . Activation of beta-mediated processes in the superficial colonic epithelium may play a role in the preventive effects observed in females and ERT users [73] . AR expression is related to the clinical stage of colon cancer [58] . Despite sex differences in tumour location and aggressiveness, most scientific researchers do not consider sex specificity in their study design and interpretation. Colorectal cancer screening guidelines do not distinguish females from males, which may explain the higher frequency of more advanced neoplasia when tumours are first detected and false negative results in colonoscopy in females [76] . Clinical Implications And Hormonal Therapy The role of estrogen in preventing and reducing the risk of CRC in vulnerable populations has been utilized as a therapeutic opportunity in CRC. Phytoestrogens (plant-derived heterocyclic phenols) and xenoestrogens (synthetic compounds) have similar binding capacity as estrogen. Xenoestrogens like industrial chemicals, dioxins, and pesticides have disruptive and negative impacts on humans. Phytoestrogens preferentially bind to Erβ, thus having a therapeutic effect on colon carcinoma [36] . Isoflavones (genistein, daidzein, glycitein, biochanin A, and formononetin), lignans (pinoresinol, lariciresinol, secoisolariciresinol, matairesinol, and enterolignans), and coumestans (coumestrol, wedelolactone, plicadin) are different types of phytoestrogens, with isoflavones being a common source of phytoestrogens in the Asian population and lignans being a common source in the Western population [79, 80] . Based on a dose-response analysis of isoflavones, the risk of CRC is decreased by 8% by increasing the intake by 20mg/day in the Asian population [79] . There is a huge difference in the intake and metabolism of isoflavones among Asian and Western populations. On average, the Asian population consumes takes <1mg/day [79] . Phytoestrogens do not produce any side effects associated with estrogen intake as they have a greater binding capacity to Erβ than Erα [80] . Intestinal flora metabolizes phytoestrogens to secondary metabolites that have a high binding capacity to Erβ. According to Grosso G et al, dietary intake of isoflavones does not lower the risk of CRC in prospective studies, but the risk is reduced in case-control studies [81] . This heterogeneity is due to the difference in the duration of isoflavone intake. According to a Women’s Health Initiative (WHI) study published in 2002, there was a significant decrease in the incidence of CRC in women using estrogen plus progestin. The study indicated a reduction of CRC risk by 33% in women taking hormone replacement therapy (HRT) [82, 83] . According to Newcomb and colleagues’ study, there was a significant reduction in CRC risk among women using postmenopausal hormone therapy and the risk reduction was more pronounced in women with prolonged use of HRT [83] . Though there are good advantages of HRT in reducing the risk of CRC, this comes with the adverse side effects of HRT in postmenopausal women. Obesity and smoking mask the benefit of HRT in lowering the risk of CRC as obesity produces a state of hyperinsulinemia that activates PI3K and increases the risk of CRC, and smoking increases the risk of MSI-linked colon cancer. Future directions Based on our study, we suggest that targeting sex hormones, either individually or in combination, holds significant potential for both the treatment and diagnosis of CRC. Future management possibilities for colorectal cancer (CRC) related to estrogen and its receptors involve the development of pharmacological agents that selectively target ERβ, harnessing the potential of selective estrogen receptor modulators (SERMs) to tailor therapies, investigating the role of the gut microbiome in CRC prevention and considering interventions like diet and probiotics, exploring hormone replacement therapy (HRT) with ERβ for postmenopausal women to reduce CRC risk, utilizing ERβ concentration as a prognostic biomarker for CRC, researching the role of ERβ in mitigating chronic inflammation, examining the relationship between estrogen, oxygen levels, and hypoxia in CRC development, studying GPER stimulation and its impact on colonic motility and constipation [25] . There is some heterogeneity found in many studies about the relationship between HRT and colon cancer risk reduction. This area needs to be addressed. Cytokines within the tumour microenvironment can also interact with oestrogen signalling, creating a complex feedback loop. Moreover, ERβ’s influence on the gut’s microbiota composition, altering local inflammation and tumorigenesis, adds another layer of intricacy. Collectively, these findings paint a dynamic picture wherein oestrogen, ERs, inflammation, and immune responses intersect, influencing colorectal cancer development and gender-associated immune variations [43] . Phytoestrogens (plant-derived heterocyclic phenols) and xenoestrogens (synthetic compounds) have similar binding capacity as oestrogen. Xenoestrogens like industrial chemicals, dioxins, and pesticides have disruptive and negative impacts on humans. Phytoestrogens preferentially bind to Erβ, thus having a therapeutic effect on colon carcinoma [36] . Isoflavones (genistein, daidzein, glycitein, biochanin A, and formononetin), lignans (pinoresinol, lariciresinol, secoisolariciresinol, matairesinol, and enterolignans), and coumestans (coumestrol, wedelolactone, plicadin) are different types of phytoestrogens, with isoflavones being a common source of phytoestrogens in Asian population and lignans being common source in western population [79, 80] . Based on a dose-response analysis of isoflavones, the risk of CRC is decreased by 8% by increasing the intake by 20mg/day in the Asian population [80] . Mahbub et al. propose that the simultaneous activation of Estrogen Receptor Beta (ERβ) and Progesterone Receptor (PGR) by their respective ligands may trigger a series of events with anti-cancer properties. This includes inhibiting cancer growth through the Androgen Receptor (AR) and Estrogen Receptor Alpha (ERα) pathways, as well as promoting anti -proliferative and pro-apoptotic effects in colorectal cancer. They propose that employing E2 and/or P4 as therapeutic agents could provide alternate approaches to combating CRC, and the efficacy of these therapies may be dependent on the expression patterns of ERs and PGR in malignant colonic tissues. Furthermore, they propose that a sequential hormone therapy involving E2 followed by P4 could be an effective regimen for early-stage CRC. In contrast, their concurrent combination may be more appropriate for advanced or metastatic colon cancer [25]. It is also noteworthy that activation of Androgen Receptor (AR) through testosterone-HSA conjugates induces apoptosis, providing a safeguard against colorectal cancer (CRC). Directing efforts towards modulating this pathway may hold the potential to improve the efficacy of therapeutic interventions for CRC patients [67] . Investigating the role of ER targeting in CRC treatment entails investigating how distinct gene alterations within tumours affect the response to estrogen-based therapies. Certain alterations may make tumours more or less responsive to these treatments, necessitating customized approaches to maximize their efficacy. Furthermore, it is critical to consider the involvement of ERs in MSI-high CRC patients. Investigating how MSI status impacts estrogen medication response can help guide decisions about its use in specific patient populations, perhaps unlocking more effective treatment regimens. Personalized medicine provides the most effective approach for patients. All of the targeted therapy plans provided for CRC allude to the link of essential genes with the central mechanisms of disease progression, all of which could be inhibited by targeted medications to prevent proliferation and invasion. However, further study is needed to investigate medication resistance mechanisms in varied groups of CRC to determine the likely prognosis in different categories of patients. Chemotherapy formulations have been viewed as increasingly challenging in recent years. Because CRC patients’ prognosis is currently confined to limited information based on the high or low frequency of MSI, the patient’s MSS status, and mutations in BRAF or PIK3CA genes, all of which influence the identification of the potential tumours’ invasion rate and differentiation, good or poor prognosis, the degree of progression, and, in some cases, the tumour’s location. Understanding how ER targeting responds differently depending on tumour location can assist in establishing the appropriate treatment methods for distinct anatomical locations, hence improving therapeutic outcomes [84] . Furthermore, there is an urgent need to discover new biomarkers that predict responsiveness to estrogen-based therapy. Creating biomarkers for ER function or estrogen responsiveness can improve treatment planning, allowing for more precise prognosis and customized therapeutic actions. Conclusion In conclusion, the cohort study provides crucial insights into thintricate world of sex steroid receptors in colorectal cancer. Altered expression patterns of ERα, ERβ, PGR, and AR in malignant specimens highlight their dynamic roles, influencing. cell cycle, apoptosis, and tumour progression. Gender-specific variations and the influence of menopausal status underscore the complexity of hormonal receptor dynamics. The correlation with clinical stage and tumour location suggests potential implications for cancer screening and treatment strategies. The findings emphasise the need for considering sex-specificity in colorectal cancer research and clinical guidelines, shedding lighon avenues for personalised interventions based on sex steroi receptor profile. Estrogen’s intricate modulation of gene transcription through ERα, ERβ, and GPER in colorectal cancer highlights the dynamic interplay of these receptors in tumorigenesis. The opposing effects of ERα and ERβ, coupled with the regulatory role of GPER, underscore the complexity of estrogen signalling. Meanwhile, the progesterone receptor’s contradictory role in colorectal carcinoma, with potential therapeutic implications, adds a layer of intricacy. The conflicting findings in progesterone receptor studies emphasize the need for standardized methodologies in assessing its impact on colorectal cancer prognosis. In conclusion, the comprehensive exploration of androge receptors (AR) and progesterone’s therapeutic potential in colorectal cancer (CRC) shed light on crucial aspects of hormonal signalling in CRC. The activation of AR, particularly its association with testosterone-HSA and the role of CAG repeats, provides insights into potential protective mechanisms and survival outcomes. Progesterone emerges as a promising monotherapy, hindering cell proliferation, inducing apoptosis, and modulating key pathways like JNK and GADD45α. These findings underscore the potential clinical implications, suggesting new avenues for targeted therapies in CRC treatment. This research enhances our understanding of hormonal influences in CRC, paving the way for refined clinical strategies and stimulating further exploration into tailored interventions and prognostic markers. *Author Contributions S.S.B., S.G., I.E., Y.J., S.S.O., K.C., R.P., S.S., and V.R.S. contributed to various sections of the manuscript, including its design, writing, and critical review. All authors have read and approved the final manuscript and agree to be accountable for all aspects of the work, ensuring accuracy and integrity. Funding This research received no external funding. Institutional Review Board Statement Not applicable, as this narrative review does not involve human or animal subjects. Informed Consent Statement Not applicable, as this study does not involve human subjects. Data Availability Statement No new data were created or analyzed in this study. Data sharing is not applicable to this article. Acknowledgments Not applicable Conflicts of Interest The authors declare no conflicts of interest. Abbreviations NENF : Neuron-Derived Neurotrophic Factor, P4 : Progesterone, PGR : Nuclear Progesterone Receptor, PGRMC1 : Progesterone Receptor Membrane Component 1, mPRα : Membrane progesterone receptor alpha, mPRγ : Membrane progesterone receptor gamma [85] EREs : Estrogen Response Elements, AP1 : Activating Protein-1 Complex, Sp1 : Stimulating Protein 1, NFκB : Nuclear Factor-κB, ER : Estrogen Receptor, c-Jun : Component of AP1, c-Fos : Component of AP1 [32,40]. REFERENCES: 1. Xi, Y., & Xu, P. (2021). Global colorectal cancer burden in 2020 and projections to 2040. Translational Oncology, 14(10), 101174. doi: 10.1016/j.tranon.2021.101174 2. Islami, F., Sauer, A. G., Miller, K. D., Siegel, R. L., Fedewa, S. A., Jacobs, E. J., … & Soerjomataram, I. (2017). 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International Journal of Molecular Sciences, 24(20), 15085.https://doi.org/10.3390/ijms242015085 Supplementary Material File (crc figures.docx) Download 2.08 MB Information & Authors Information Version history V1 Version 1 02 January 2025 Peer review timeline Published Annals of Medicine & Surgery Version of Record 28 Jan 2025 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords androgen receptor (ar) colorectal cancer (crc) estrogen receptor beta (erbeta) estrogen-progesterone therapy progesterone receptor (pgr) sex steroid receptors Authors Affiliations Shreya Singh Beniwal Lady Hardinge Medical College View all articles by this author Srajan Gupta Sri Venkateswara Medical College View all articles by this author Indu Etta Kakatiya Medical College View all articles by this author Yash Janu Government Medical College Kota View all articles by this author Sidharth Olikkal S 0009-0004-6062-8139 Government Medical College Thiruvananthapuram View all articles by this author Kareema Cummings Central Library View all articles by this author Roma Patil Bangalore Medical College and Research Institute View all articles by this author Saif Syed RSCI Department of General Practice View all articles by this author Vaishnavi Rajesh Shetty Lvivs'kyj nacional'nyj medychnyj universytet imeni Danyla Halyc'koho View all articles by this author Ayush Dwivedi 0009-0005-1955-9116 [email protected] Lvivs'kyj nacional'nyj medychnyj universytet imeni Danyla Halyc'koho View all articles by this author Metrics & Citations Metrics Article Usage 1068 views 165 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Shreya Singh Beniwal, Srajan Gupta, Indu Etta, et al. 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