Sexual dimorphism refers to the differences in morphology and physiology between males and females of the same species. This concept is particularly important in the study of diseases, as it results in different susceptibilities for each sex, influencing mortality, morbidity, prevalence, intensity, severity, behavioral changes, and immune response (1). For instance, colorectal cancer (CRC) is the third most common heterogeneous neoplasm worldwide, with several studies reporting a higher incidence in men than in women (2). The causes of sexual dimorphism can be attributed to various factors, including gender and sex distinctions such as behavioral and physiological differences (3). Moreover, sexual dimorphism appears to persist regardless of ethnicity or geographic location, suggesting that intrinsic factors play a fundamental role in explaining these disparities (2).

The concentration of sex steroids is one of the most important intrinsic factors that differ between both sexes (4). These molecules play crucial roles in the gut: Estrogens influence epithelial membrane permeability, serotonin production, the expression of tight junctions, inflammation, and microbiome composition (5). On the other hand, the effects of androgens are not fully understood. However, in a mouse model, they have been shown to alter the proliferation of enterocytes and increase the size of crypts (6).

In colorectal cancer patients the levels of sex steroids are altered. For instance, postmenopausal women with CRC have higher levels of 17β estradiol (E2) and estrone and a greater ratio of testosterone (T4) to E2 than control patients do; however, this pattern is not observed in men (7). On another hand, the expression of aromatase, an enzyme responsible for converting T4 to E2, is greater in neoplastic tissue in both men and women. This expression is inversely associated with the proliferation index, tubular differentiation, and estrogen concentration just in men and not in postmenopausal women (8). Consistently, serum levels of E2 are greater in male patients with CRC than in healthy controls (9).

The relationship between androgens and CRC has been poorly studied. Contrary to expectations, the circulating level of T4 is inversely associated with overall survival and mortality in CRC patients but only in men (10, 11). A reduction in androgen levels is associated with fewer CAG repeats in the androgen receptor (AR) gene, which subsequently increases transcriptional activity (12). AR is overexpressed in colon tumor tissue and is associated with tumor size, differentiation, and distant metastasis (13). In contrast, this receptor is not expressed in the non-neoplastic mucosa. Interestingly, in postmenopausal women, a few CAG repeats in the AR gene and a CA repeat in the ER-β gene are associated with high serum androgen levels, suggesting that these polymorphisms have a stimulatory effect on T4 production in women (14). Although further research is needed to understand deregulation of sex steroid concentrations and its importance in the physophatology of CRC.

In vitro studies with colon cancer cell lines such as DLD-1, HCT-116, SW480, and CaCO2 have revealed that E2 reduces the viability of these cells, while T4 and dihydrotestosterone (DHT) increase apoptosis in CaCO2 and HCT116 (5, 15–24). E2 reduces the viability of colon cancer cells by activating P53, which subsequently upregulate the levels of p21 and p27, that consequently inhibits cyclin D1 gene that reduce proliferation (25). Additionally, the treatment with E2 decreases cellular migration by reducing MMP-2 and MMP-9 levels and inhibiting the JNK 1/2/PGE2 pathway, which is involved in cellular motility (26).

Experiments with microarrays have shown that ER-β plays an important role in the regulation of transcription factors such as MYC, MYB, RUNX2, and PROX1, which are involved in cell viability, proliferation, apoptosis, and differentiation. This modulation potentially triggers an antitumoral cascade (27). On another hand, T4 increases apoptosis in cells by reducing PI3K/Rac1, which activates the kinase JKN, stimulating the transcription of proapoptotic genes involved in intrinsic and extrinsic apoptosis (23, 28). Finally, reorganization of the cytoskeleton reduces the migration and invasion of colon cancer cells. This effect is not influenced by the inhibition of aromatase, suggesting that the effect is directly mediated by T4 and not by the conversion of T4 to E2 (29); since, this enzyme metabolizes T to E2 and E2 is a final metabolite in the steroidogenesis.

There are few in vivo studies in which sexual dimorphism is evident. Lee et al. described a rapid and effective method for modeling human colitis-associated cancer using the carcinogen azoxymethane (AOM) and subsequently treating ICR mice with the pro-inflammatory agent dextran sulfate sodium (DSS) (30). In this study, they observed that male mice developed more tumors than females; however, the role of sex steroids remains unclear. For instance, male PIRC rats developed twice as many adenomas as females (31); In this research, neither ovariectomy nor reconstitution with E2 in PIRC rats affected the development of adenomas. In contrast, Song et al. showed the protective effect of endogenous or E2 replacement in ovariectomized female ICR mice against AOM/DSS-induced colitis (32). In another research, males treated with estradiol develop fewer tumors and less tissue damage (33). In an inflammation context, there are few parameters that has been studied to explain the protector effect of E2 in both sexes. This hormone reduce inflammation diminishing parameters such as COX-2, IL-6, TNF-α, iNOS and IL-1β (32, 33).

In the AOM-DSS model in C57BL6 mice, T4 significantly enhances the induction of colorectal cancer (34); Conversely, orchiectomy in PIRC rats drastically reduces the number of tumors, and this effect is countered by reconstitution with DHT (31). In contrast, treatment with a conjugate of T4 and albumin significantly reduces tumor incidence induced by dimethylhydrazine and DSS in Balb/c mice (35). These apparently contradictory results could be explained by T4 potential metabolism into E2 and DHT, making it challenging to elucidate its specific effects. Furthermore, its conjugation with albumin primarily interacts with the androgen membrane receptor. The above findings underscore the importance of conducting a study with standardized conditions, to assess the involvement of sex steroids in the induction of colorectal tumors in both males and females. Therefore, the goal of this research is to determine whether sex-related susceptibility in the induction of colorectal tumors is mediated by the concentrations of the principal sex steroids (E2 and DHT), evaluated in a murine model.

In humans, CRC is one of the most common malignant neoplasms, ranking 2nd to 4th in terms of incidence depending on factors such as location, cancer type, or gender. The study of risk factors is highly relevant due to the increasing incidence in terms of morbidity and mortality. Several factors may be responsible for the development of this disease. The progression from adenoma to carcinoma exhibits clear sexual dimorphism, with a preference for male development of both adenomas and CRC (31). Therefore, we undertook a study to investigate the role of sex hormones in CRC progression. We used a murine model widely accepted through the AOM-DSS method. Gonadectomy was performed to evaluate the influence of sex steroids in this process, reconstituting and substituting with the principal sex steroids (E2 and DHT).

As expected, the induction of tumors showed a dimorphic pattern. Males exhibited greater growth of tumors in size and number, and a significantly higher TBS compared to females. This parameter considers the number and the maximum tumor size in the tissue. In addition, recent studies have shown that tumor burden score is a good marker for prognosis and is associated with some molecular mutations such as KRAS (41).

Consistent with our findings, previous studies involving ICR, C57BL/6 and APC/min male mice had shown a higher incidence of tumors compared to females (31, 33, 34). In our results, gonadectomy in females increased the number of neoplasias and resulted in a higher TBS than the sham group. In contrast, in males, orchiectomy reduced the number of tumors, lowered the fold change in tumors larger than 2 mm and decreased the TBS value. This demonstrates a greater effect on the fold change in the number, size, and TBS of tumors in males undergoing orchiectomy compared to females undergoing ovariectomy. This observation is particularly relevant, as not only the number but also the size of tumors and their correlation (TBS) in CRC are closely associated with prognosis, including tumor stage and metastasis.

This suggests that androgens play a more significant role in the progression of colorectal tumors compared to estrogens, at least in males (42, 43). Although DHT levels are not modified in intact males compared to control male mice, the levels of T4 were lower in control animals (with tumors). This suggests that DHT is responsible for the protumoral effect. In line with this, the administration of DHT in both sexes favored the growth of tumors compared to gonadectomized groups. Consistently with our results, orchiectomy in PIRC rats, which are spontaneously susceptible to developing adenomas, reduced the formation of neoplastic lesions, and reconstitution with DHT increased the number of tumors (31). These observations open the possibility of using drugs that inhibit the interaction of androgens with their receptor, such as flutamide or apalutamide, both of which are approved for the treatment of prostate cancer.

The decrease in T4 levels and the increase in the concentration of E2 in control males, when compared with intact mice, suggest that T4 is being converted to E2. Consequently, the serum concentration of T4 in patients with CRC is lower compared to control cases. Additionally, CRC patients exhibit a higher expression of aromatase (enzyme that convert T4 in E2) in neoplastic tissue associated with E2 in men (8, 44). Interestingly, the lower concentration of circulating T4 is associated with overall survival, although the expression of androgen receptors is directly associated with tumor size, tumor differentiation, and distant metastasis (11, 45). Unexpectedly, the concentration of DHT was higher in control females (with tumors) compared to the intact group. However, ovariectomy significantly decreases the levels of this hormone to undetectable levels. On the other hand, T4 levels are reduced in ovariectomized females, suggesting that T4 is being converted to DHT. Although the substitution with DHT increases the number of tumors by 1.4 times compared to Gx females, the concentration of DHT is higher compared to control animals. This suggests that the biological effect on tumor growth depends on the dose. Another possible explanation is that E2 is the principal factor that plays a role in the development of colorectal tumors in females.

Ovariectomized females had lower levels of E2 compared to intact and control mice. Ovariectomy increased the number of tumors and the TBS relation. Reconstituted females exhibited a decrease in the number of tumors and the TBS value. These findings suggest that estrogens play an antitumoral role, in alignment with epidemiological, in vitro, and in vivo studies. For instance, hormonal therapy replacement and the consumption of foods rich in phytoestrogens reduce the risk of developing CRC in postmenopausal women. Unfortunately, HTR is associated with a higher risk of breast cancer (46–48). Estrogens favor the proliferation and mutation of breast cancer cells by the interaction with the estrogen receptor alpha (48). In contrast, the expression of estrogen receptor β is directly associated with high survival and inversely with the stage of tumor in CRC patients (49). The above support the idea of promoting the use of specific agonists of estrogen receptor β such as diarylpropinitrile that inhibit the viability of colon tumor cells in vitro.

On the other hand, the substitution with E2 increased the number and size of tumors, as well as the TBS, when compared to Gx animals. This suggests a dimorphic mechanism of action for this hormone. Additionally, CRC patients have lower levels of T4, and increased aromatase levels result in higher intratumoral E2 concentrations. Overexpression of aromatase is associated with adenocarcinoma differentiation. More studies are necessary to fully elucidate the dimorphic role of E2 in males and females. It is important to mention that in this study, we evaluated a single dose for reconstitution and hormonal replacement. This leads us to think that the dimorphic effect of E2 could depend on the concentration and form of administration. In other studies with C57BL/6 male mice, this hormone had a protective effect when administered intraperitoneally at a very high dose (10 mg/kg every 7 days) (33). On the other hand, in a female PIRC rat model that develops tumors spontaneously, estradiol had no effect on tumor development when administered via slow-release pellets at a low dose (∼4 μg/kg/day) (31). This hormone could be modulating the tumor microenvironment differently as its effect on target cells depends on the dose. For instance, at high doses, E2 generally has an immunosuppressive effect, whereas low doses promote the production of proinflammatory cytokines (50). Interestingly, aromatase (the enzyme that converts T4 to E2) is overexpressed in the colon of men with colorectal cancer compared to healthy individuals (8). Furthermore, the expression of alpha estrogen receptors increases in tumor tissue compared to normal mucosa in men but not in women (7). The expression of this receptor is associated with a poor prognosis in CRC patients (51). This leads us to consider that another explanation for the dimorphic effect of estradiol, besides its concentration, is that it depends on receptor expression in the tumor. For example, in breast cancer, estrogen receptor beta is poorly expressed in breast tumors; however, it is associated with better prognosis and reactivation of the anti-tumor immune response (52). Activation of this receptor inhibits the cell cycle and also reduces the formation of angiogenic factors (53).

Regarding the mechanism, in vivo, E2 affects important pathways associated with inflammation and colonic tumor development such as NF kB, NRF2 and NLRP3. In vitro, the study with different tumor cell lines has shown the activation of pathways such as MAPK and Wnt/β-Catenin through the β estrogen receptor that inhibit the proliferation of tumor cells (33, 54).

On the other hand, in vivo, T4 increases the expression of some inflammatory factors such as MPO, IL-1, COX2 and INOS that are associated with the development of colorectal cancer. In vitro, this hormone modulates the Akt pathway, influencing cell adhesion and migration, either promoting or inhibiting it through the intracellular or extracellular androgen receptor, respectively (34, 55).

In our study, we observed no significant effect on the progression of colon cancer cell lines when treated with DHT. This suggests that the impact of these hormones might be mediated through their interaction with other cells in the tumor microenvironment, such as immune cells, stromal cells, nervous system cells, or microorganisms in the intestinal microbiota. On the contrary, our results indicate that E2 inhibits the proliferation of HCT116, a cancer cell line derived from males. Interestingly, this hormone promotes tumor growth in male animal models. It would be important to conduct a broader evaluation of the effect of estradiol and dihydrotestosterone on other cell lines derived from tumors of both men and women, as well as from non-neoplastic tissue. There are no studies in the literature on cell lines with this dimorphic focus. Additionally, this focus could help us begin to understand the discrepancies between the in vitro analyses conducted in this publication and the in vivo results that we obtained. On another hand, it would be worthwhile to investigate the effects of sex in other systems, such as the production of cytokines, chemokines, and neurotransmitters that are so important in the microenvironment of colon tumors.

Recently, our group conducted a review that specifically addresses the role of sex steroids in the pathophysiology of CRC, focusing on their effects on immune system cells, neurons, and their soluble factors (neurotransmitters and cytokines) (56). All these factors interact within the tumor microenvironment and play a significant role in the development of colorectal tumors (56). Our study revealed that E2 has an anti-inflammatory effect on the innate immune response, especially in studies that directly assess inflammation in colonic epithelium tumors. However, it is important to mention that estrogens can activate dendritic cells and lymphocytes, which are essential cells for tumor elimination, and their infiltration into the microenvironment is clearly associated with a favorable prognosis. E2 affects the enteric system. Primarily, it stimulates neurogenesis and maintains tissue architecture. Damage to enteric glial cells is a consequence of cancer invasion, and this damage leads to the release of prosurvival and proliferative signals that create a microenvironment favorable for tumor growth. Furthermore, although the effect of the microbiota is complex, E2 increases bacterial diversity, which is associated with a good prognosis in patients with colorectal cancer and reduces tumor progression in animal models by their interaction with ER-β. Apart from CRC, to our knowledge there are no studies that dimorphically study the effect of estradiol on other types of cancer (56).

Conversely, androgens play an immunosuppressive role in both the innate and adaptive immune systems. These findings suggested that these molecules help tumors evade immune responses. Additionally, although studies suggest that androgens do not alter neurogenesis, they can stimulate the synthesis of molecules such as those in the sympathetic nervous system (adrenaline and noradrenaline), 5-HT, Ach, and their receptors. These molecules promote tumor cell proliferation, tissue degradation, tumor formation, and the production of angiogenic factors such as MM-9 and 3. Furthermore, they stimulate the secretion of IL-12, IL-17, and IFN-γ, which potentially promote carcinogenesis through chronic inflammation. In addition, males have a lower diversity of microbiota than females, and animal models suggest that androgens favor a decrease in biodiversity, while the microbiota increases the bioavailability of androgens in the colon. Decreased diversity is a risk factor associated with poor prognosis in the early stages of CRC carcinogenesis (56).

In other types of cancer, we can observe the clear pro-tumoral role of androgens in the development of these diseases. Epidemiologically, men have a higher risk of almost all non-sex-dependent cancers. For example, high levels of T4 and DHT are associated with an increased risk of developing lung cancer (57). Moreover, androgen receptor knockout mice develop smaller tumors, and these hormones polarize lung macrophages toward an M2 phenotype that enhances tumor growth through immune suppression (58). Another example is seen in a mouse model of bladder cancer, where the percentage of males with tumors is higher than females, and DHT increases tumor growth and lung metastasis in males (not tested in females) (59). Androgens also play a significant role in breast cancer, traditionally associated with estrogens. In this cancer, they play a dual role by increasing proliferation in estrogen receptor-positive breast neoplastic cells and promoting it in triple-negative tumor cells (60). These examples underscore the importance of studies evaluating the role of androgens in tumor development in both sexes. Furthermore, it’s important to highlight their generally immunosuppressive effect, suggesting they prevent the immune system from eliminating tumors (61).

Epidemiological analysis show that sex is a statistic risk factor associated with CRC in men. Our results indicates that androgens have a protumoral role while estradiol have a dimorphic effect. In females this hormone protects and in males stimulate the development of tumors. The molecular and cellular mechanisms are not dilucidated in this work. However, our group is working to analyze the effect of sex steroids on the tumor microenvironment that involves cytokines, chemokines, neurotransmitters and microbiota.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

All experimental procedures in the animals were approved by the Institutional Care and Animal Use Committee (Full name: Consejo para el Cuidado y Uso de Animales de Laboratorio, CICUAL), adhering to Mexican regulation (NOM-062-ZOO-1999), in accordance with the recommendations outlined in the National Institutes of Health (NIH) of the United States of America (Guide for the Care and Use of Laboratory Animals). The animal care and experimental practices were conducted at Unidad de Modelos Biológicos (UMB) in the Instituto de Investigaciones Biomédicas (IIB), Universidad Nacional Autónoma de México (UNAM). All experimental procedures in the animals were approved by the CICUAL, ID Number 6298. The present study adheres to the ARRIVE guidelines. For tumor induction, we utilized the gold standard protocol for inducing colorectal tumors (check material and methods). Throughout the study, the animals were monitored daily to promptly identify and alleviate signs of pain or distress.

YR: Formal analysis, Investigation, Methodology, Visualization, Writing – original draft. LT-V: Conceptualization, Formal analysis, Supervision, Writing – original draft. KN: Data curation, Formal analysis, Funding acquisition, Investigation, Resources, Writing – original draft. VD: Investigation, Validation, Visualization, Writing – original draft. CG: Investigation, Methodology, Project administration, Visualization, Writing – original draft. JM: Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review & editing.