Role of αvβ3 Membrane Receptor, as a Thyroid Hormone Receptor, in Liver and Colon Cancer Induction

IntroductionThyroid hormones (THs) include thyroxine (T4), triiodothyronine (T3). These hormones affect cell growth and differentiation. Increased basal metabolic rate is the main effect of thyroid hormones. Binding of THs to αvβ3 integrin facilitates the proliferative function of hormones on cancer cells and also angiogenesis. The integrin αvβ3 with their receptors are also associated with fibroblast growth factor 2 (FGF2), epidermal growth factor (EGF), matrix metalloproteinase-2 (MMP-2), metalloproteinase.

Conclusion Therefore, the aim of this study was to investigate the role of αvβ3 receptor in development and progression of cancer in liver and colon tissues. Our study showed that the expression of avβ3 is associated with tumors progression in liver and colorectal cancer. Furthermore, here we emphasized the capability of αvβ3 in diagnosis and prognosis of liver and colorectal cancer. Finally, determination of αvβ3 receptor levels seems to be used as a prognostic factor in colon and liver cancer.

Keywords: αvβ3 membrane receptor; thyroid hormone receptor; thyroxine; triiodothyronine; liver cancer; colon cancer

In recent decades, the incidence of cancer as well as deaths has grown dramatically, around the world. Gastrointestinal cancer is the fifth most common cancer and the fourth leading cause of cancer death, in both sexes. According to the American Cancer Society in 2020, colorectal cancer (CRC) ranked third in incidence but is second in mortality[1]. Metastasis is the leading cause of death from gastrointestinal cancer [2].

Although many attempts have been made to identify the biochemical mechanisms involved in the development of liver and colorectal cancer, due to their complexity, these mechanisms remain obscure. In these ambiguous conditions, the identification of genes that metastasize gastrointestinal cancers, effectively, helps to identify new mechanisms and therapeutic goals. New therapeutic targets in tumors and a wide selection of anticancer drugs have more effectively improved treatment strategies for CRC and hepato carcinoma in recent years, which have played a vital role in patient well-being [3].

More efficient surgical techniques to control metastatic liver and colorectal cancer have improved the overall survival of CRC patients. Also a combination of 5-fluorouracil (5-FU), folinic acid, fluorouracil, oxaliplatin (FOLFOX) - and folinic acid in CRC patients increased the survival rate from 14.2 to approximately 30 months [4]. However, this improvement did not increase the 5- year survival rate for patients with end stage disease [5].

THs act through a set of well-studied nuclear receptors called thyroid hormone receptors (THRs) [6]. THRs along with other molecules, attach to specific areas of DNA called thyroid hormone response elements (THREs) near the genes. This complex, if combined with corepressors, can block transcription of genes [7]

Several studies have reported the stimulatory effect of thyroid hormone on the growth and proliferation of cancer cells [8], and in some clinical studies of patients with hypothyroidism, reduction of tumor growth in cancer patients has been shown [9].

A study has shown that thiouracil-induced hypothyroidism delays the growth and reduces the incidence of breast tumors in mice [10]. This study reported that hypothyroidism can contribute to localized atrophy of the mammary glands, thereby reducing the growth of tumor tissue. But the results of the other study were so contradictory that in the same study, hyperthyroidism reduced tumor growth [13]. Other study reported that Graves' disease appears to be associated with thyroid cancer. Patients with diffuse goiter are also at high risk for thyroid cancer [12]. The results of studies on the role of hyperthyroidism and hypothyroidism with colon cancer are highly contradictory. Some studies have shown that hypothyroidism is associated with a lower risk of colorectal cancer [13], while other study showed that the hyperthyroidism is linked with a lower risk of colon cancer [14]. Although numerous epidemiological studies showed association of thyroid dysfunction with liver cancer and CRC, But there are many contradictions in the relationship between liver cancer and thyroid hormones, so TRα mutations in hepatocellular carcinoma cells act as dominant negative inhibitors and disrupt gene transcription [15]. In contrast to these results, TRβ mutants can have negative effects. Consequently, TR mutations may have roles in cancer development [15].

Integrins are a group of plasma membrane proteins that bind to and interact with extracellular matrix (ECM) proteins such as vitronectin, fibronectin, and osteopontin, and their key functions is to regulate cell adhesion [16]. Both thyroid hormones bind to the αvβ3 integrin, but it is important to note that T3 binds to the S1 receptor site and activates PI3-K. The second site is S2 that leads to activation of the MAPK pathway and cell proliferation [17]. Integrin αvβ3 is involved in angiogenesis and tumor metastasis [18]. The integrin αvβ3 is also associated with FGF2, MMP-2, and VEGF [19], which inhibits apoptosis and accelerate cell proliferation [20]. Also previous studies noted that inhibition of αvβ3 integrin induces cellular apoptosis and inhibits angiogenesis [21]. The vital role of αvβ3 integrin in tumor angiogenesis has raised new therapeutic hopes that by inhibiting this receptor (αvβ3 integrin), the cell proliferation process stops and the cell enter the apoptotic phase.

Based on this background, the aim of this study was to investigate the role of αvβ3 receptor in the development and progression of cancer in liver and colon tissues.

Mechanisms regulating gene expression cause a series of molecular changes in DNA and chromatin and cause different physiological signals in the cell. Regulation of gene expression is controlled by specific regulators, each of which binds to specific sequences in DNA and produces proteins that are responsible for structural changes in chromatin [22]. Chromatin contains double-stranded DNA and proteins whose main unit is the nucleosome. Chromatin contains euchromatin, which is the active part of DNA for transcription, and heterochromatin, which is the inactive part of DNA [22]. Various forms of histone acetyltransferase (HAT) and histone deacetylase (HDAC) have been identified, in addition to other coactivators and corepressors that can interact with transcriptional regulators [23]

THRs are available in both alpha and beta forms. The location of these subtypes largely depends on post-transcriptional changes. THRs genes are located on chromosomes 3 and 17. There are three types of THR-α receptor encoded by the THRα (alpha thyroid hormone receptor) gene and three types of TR-β isoform encoded by the THRß (beta thyroid hormone receptor) gene. T4 is only able to bind to four of them: TR-α1, TR-β1, TR-β2 and TR-β3 [8]. When T3 binds to its receptor, it causes a structural change in the receptor and displaces the corepressors from the complex. This complex binds to the RNA polymerase, which activates gene transcription [8]. Nuclear Co-Repressor (NCoR) and silencing mediator of retinoic acid (SMRT) are the key regulators of nuclear receptor signaling [30]. NCoR and SMRT interact with THRs isoforms. Hypothesized NCoR and SMRT regulate Triiodothyronine (T3) target genes in the absence of ligand Triiodothyronine (T3 ) [24]. Therefore, NCoR and SMRT are involved in the pathophysiology of hypothyroidism [24].

Retinoid X receptor (RXR) is a nuclear receptor activated by 9-cis retinoic acid that interacts with THRs [25]. There are three retinoic X receptors RXR: RXR-alpha, RXR-beta, and RXR-gamma. RXR is heterodimerized with subfamily 1 nuclear receptors including peroxisome proliferator activated (PPAR), retinoic acid (RAR), THR, and vitamin D receptor (VDR).

Nuclear receptors of THRs have a zinc-rich structure that can bind to double-stranded DNA (DNA Binding Domain). THRs bind to TREs containing the 5 '- (A/G) GGT (C/A/G) A-3' nucleotide sequence [26]. When THR binds to T3 , the C-terminal ligandbinding domain (LBD) undergoes a structural change that allows it to interact with coactivators [27]. T3 acts as a major mediator in the metabolism of thyroid hormones by forming complexes with alpha-nuclear THRα and THRβ. These specific T3 nuclear receptors bind to thyroid hormone response elements and regulators the transcription of metabolism-related genes [28]. THRs bind to corepressors that suppress gene transcription. When T3 binds to receptors, corepressors are released and transcription coactivators transcript the gene expression process and T4 or T3 stimulates the growth and proliferation of cancer cells. A coactivator is a type of transcriptional coregulator that binds to an activator (a transcription factor) to increase the rate of transcription of a gene or set of genes. Coactivators and corepressors are a type of transcriptional regulator that binds to an activator or a repressor (a transcription factor) to increase or decrease the rate of transcription of a gene or set of genes [29]

Non-genomic or non-transcriptional activity of thyroid hormone occurs at a specific receptor on the cell surface of a protein called αvβ3 integrin or in truncated THRα isoforms in the plasma membrane or cytoplasm [30]. Studies previously showed that the non-genomic effects of thyroid hormones were exerted only by non-nuclear receptors, but recent evidence has shown that non-genomic effects are mediated in the cytoplasm by nuclear receptors [31].

Molecule avβ3 integrin can also bind to other small molecules such as resveratrol and steroid hormones (estrogen and androgens), and cancer growth may be modulated by this type of interaction and studies demonstrated that resveratrol induced apoptosis on cancer cells [28]. These receptors are made of a separate peptide. The peptide has three distinct functionalities: the domain at the amine terminus, the domain binding to the central DNA, and the domain binding to the terminal ligand located in the carboxyl region. Terminal domain ligand provides the conditions for the formation of homodimers or heterodimers with other members of the nuclear receptor superfamily [32]. The αvβ3 integrin has been introduced as a vitronectin receptor, consisting of a αv125 subunit with 125kDa and a 105 kD β3 subunit. It is noteworthy that αvβ3 binds to different types of molecules that have the specific structure of three ArgGly-Asp (RGD) peptides present in the molecules of fibronectin, fibrinogen, von Willebrand factor, vitronectin [33,34]. The major mediators of the non-genomic function of thyroid hormone on metabolism are protein kinase signaling cascades. In other words, the pathway by which thyroid hormones induce cancer in liver and colon is through the activation of kinases that activate growth factors [34]. This cascade lead to the movement of the phosphatidylinositol-4,5- bisphosphate 3-kinase (PI3K) into nucleus for expression of genes: Hypoxia-inducible factor 1-alpha (HIF-1α) and Na+ /H+ exchanger in the plasma membrane. PI3K is one the most important factors that activated by THs in human endothelial cells [35]. Increased Na+ /K+ATPase function of plasma membranes is mediated by PI3K/AKT/PKB [36]. Ca2+-.H+ ATPase pump activity is also mediated by local non-genomic function of thyroid hormone through stimulation with T3 . Also, the S2 fragment in αvβ3 binds preferentially to T4 and activates the specific protein kinase by (MAPK or ERK1/2) [37]. The metabolic result of T4 binding to the S2 fragment in αvβ3 causes cell proliferation and increased angiogenesis [21]. Studies have shown that αvβ3 integrin is transported into cells through cell-dependent mechanisms in the cell membrane [38]. One mechanism that has been introduced is that the αvβ3 integrin is phosphorylated and also binds to caveolin during endocytosis [39]. In the next step, β-integrin is separated from its complex and only the α-caveolin integrin complex binds to ERK1/2 protein [40]. In the next step, the αvERK1/2 complex is transferred to the cell nucleus and plays a role in the transcription of specific genes by binding to other transcription factors, and cancer cell and tissue proliferation increases [41]. Another non-genomic function of thyroid hormone is that one of the isoforms of THRα outside the nucleus conserves actin cytoskeleton by T4 and rT3 . Another THRα isoform called p30 in plasma membranes is regulated by T3 to regulate non-malignant cell proliferation through the ERK and AKT pathways [37].

THRβ is also known as the nuclear receptor subfamily 1, group A, member 2 (NR1A2), a nuclear receptor protein encoded in humans by the THRß gene [31].

The complex of THRβ and T4 is transported into the nucleus and binds to specific coactivators and plays a role in transcription of growth factors [42]. Evidence suggests that traditional THRβ-T3 plays a negative role in cancer cell proliferation [43]. THRβ plays an important role in all cellular activity regulated by thyroid hormone [36]. Studies have shown that THRβ_T4 or T3 complex plays a role in suppression of cancer. THRβ shows anti-proliferative effects in cancer cells. Importantly, loss of gene expression or decreased THRβ function is associated with the development of cancer. It should also be noted that THRβ is involved in the growth and development of cancer if it has a mutation [37].

THRα plays a dual role in activating or suppressing its target genes. Studies have shown that THRα, which is located in the nucleus attached to DNA, is moving between the nucleus and the cytoplasm [44]. The tissue distribution of thyroid hormone receptors is not the same in different tissues and THRα-1 has the highest expression in bone, digestive system, cardiac and skeletal muscle and central nervous system. THRα2 and THRα3 in the brain, kidney, testicle, brown fat tissue and skeletal muscle are dominant. THRα2 is found in mammals but has not been observed in non-mammalian species. There is enough evidence that THRα2 modulates the expression of thyrotropin-releasing hormone (TRH) gene in the hypothalamus. In addition, there are four shortened forms of THRα1 receptor, which include p33, p43, p30, and p28. It should be mentioned that shortened forms of the THRα receptor, i.e. THRΔα1 and THRΔα2, are also present in the tissues, which are mostly expressed in the brain, lung and digestive system [44]. THRα-1 bound to T3 is the major isoform for this binding. Whereas THRα-2 does not have the capacity of T3 binding and instead acts as an antagonist for T3 signaling [45]. THRα-2 is positively associated with lower mortality in cancer [45]. Binding of T3 to THRα-1 increases phosphorylated AKT levels in liver cancer, and phosphorylated AKT is overexpressed in several cancers. It is also associated with a worse prognosis in liver and colon cancer [46].

The αvβ3 protein is a plasma membrane integrin that is a membrane receptor for TH [16]. Several study showed that integrin avβ3 is involved in angiogenesis and tumor metastasis [47,48]. Binding of thyroid hormones to αvβ3 integrin facilitates the proliferative function of hormones on cancer cells [28]. The role of T4 in cancer cells begins with binding to the αvβ3 integrin [59]. Tetraiodothyroacetic Acid (Tetrac) is a derivative of T4 . This molecule competes closely for binding to αvβ3 integrin (T3 and T4 ) and blocks the carcinogenic activity of these hormones in cancer cells. Tetrac, a thyroxine analogue, prevents the proliferation of thyroid hormone-dependent and metastatic cancer cells. The main mechanism that should be noted in colorectal cancer is high expression of β-catenin genes [50]. Wnt/β-catenin signaling has a key evolutionary pathway in embryonic development and tissue homeostasis. Any defect in this pathway contributes to the occurrence of diseases. Improper activation of this pathway causes β- catenin to accumulate in the cell nucleus and causes transcription of many oncogenes such as CyclinD-1. As a result of this increased transcription, there are conditions for carcinogenesis and the growth and development of tumor tissue, including colon cancer [51]. The Tetrac molecule increases expression of chibby family member 1 (CBY1), which is a β-catenin antagonist, thereby stopping cell proliferation [52].

Integrin avb3 is highly expressed in cancer cells and dividing endothelial cells. Integrin avb3 controls a variety of intra-cellular and trans-cellular functions. The effects of T4 and T3 on gastrointestinal cancer cells are different. For example, in gastric cancer, the amount of T3 probably accumulates in gastric cells [53]. This increase of T3 inside gastric cells induces the expression of HIF1α and contributes to cancer progression, and also activates the expression of VEGF, which plays a key role in angiogenesis. It should be noted that the effects of T3 are mediated by the accumulation of fumarate that inhibits HIF1α degradation. These effects of T3 are mediated through PI3K signaling [53]. Acting on αvβ3 receptors, T4 stimulates colon cancer cell proliferation and activation of PCNA, cyclin D1, and c-Myc. These precancerous effects of T4 can be prevented with tetrac and nanotetrac [52]. These promitogenic effects of extracellular T4 are in sharp contrast to mechanisms initiated by T3.

Integrin αvβ3 serves is a receptor for extracellular matrix proteins with sequence arginine-glycine-aspartic (RGD). Integrin αvβ3 is expressed at low levels on epithelial cells and [54] mature endothelial cells; but it is highly expressed on activated endothelial cells in neovasculature of tumors, including osteosarcomas, neuroblastomas, glioblastomas, melanomas, and several cancers [55]. RGD peptides that bind to the αvβ3 integrin as revealed by the crystal structure of the αvβ3 ectodomain in combination with the cyclic RGD peptide – cilengitide [4] – provide the structural basis for the development of αvβ3 antagonists. Targeting tumor cells or tumor vessels with RGD-based strategies is a promising approach to deliver anticancer drugs or contrast agents for cancer treatment and diagnosis. RGD-based strategies including RGD peptide or peptidomimetic drugs, RGD compounds, and RGD peptide or peptidomimetic grafts have been introduced in cancer therapy [56]. The vital role of αvβ3 integrin in tumor angiogenesis has raised new therapeutic hopes that by inhibiting this receptor, the cell proliferation process stops and the cell enter the apoptotic phase [57].

The effect of TH signaling on hepatocytes and hepatocellular carcinoma (HCC) has been investigated in several studies. Studies have shown that T3 causes the proliferation of liver cells in cell culture conditions and animal models [58]. Treatment of mice with T3 induced the expression of the key factor VEGF in hepatocytes, which is the role of T3 in angiogenesis [59]. THRβ is the most abundant receptor in liver tissue, and it seems that this receptor has an important role in the development of HCC. TRHβ1 expression caused the proliferation of cancer cells in human HCC cell lines [60]

Liver fibrosis and cirrhosis are characterized by chronic damage to the liver tissue, which leads to chronic inflammation and changes in the liver tissue and vessels, and in this inflammatory condition, the liver loses its function and eventually lead to become cancer [61]. Alterations in thyroid function may contribute to the development of liver fibrosis especially in subjects with non-alcoholic fatty liver disease. Low-normal thyroid function is associated with higher greater risk of liver fibrosis in the general population, being dependent on other metabolic factors [62].

A number of proliferative activators in hepatocytes are also known, including transforming growth factor beta (TGF-β) and integrins, which enable communication between ECM proteins and cells. TGF-β is a cytokine that is particularly associated with tumor progression [62]. TGF-β has been shown to affect tumor cells to regulate tumor progression [63]. TGFβ has a dual role in cancer and is both a tumor suppressor and a stimulant of tumor metastasis. Tumor suppressing effects of TGFβ are seen in normal and cancer cells through inhibition of cell proliferation, induction of apoptosis. Conversely, the tumorigenic effects of TGFβ, which include cell adhesion, migration, invasion, absorption chemistry, and tumor metastasis, are particularly observed in invasive and invasive tumors [64]. Most cancer cells lose the ability of TGF-β to inhibit growth [65]. Thyroid hormones are important modulators of the regeneration process, as they can interact with growth factors such as EGF and TGF-β as well as integrins [66].

In liver cancer cells, T3 is a mitogen that acts by activating protein kinase A (PKA), thereby activating cyclin D1 then apoptosis inhibited [53]. Of course, it is notable that hyperthyroidism is also associated with cancer, although there are contradictory results in this regard. Decreased expression of nuclear THRs acts as a signal for anti-tumorigenesis. Therefore, the conversion of hyperthyroidism to hypothyroidism can be introduced as an antitumor agent [68]. Also, the relationship between thyroid hormones and variants of receptors seems to be important in the progression or inhibition of liver cancer growth [69]. There is conflicting information in vitro about the effect of THs on hepatocellular cancer (HCC). Several studies have reported that T3 binds to its receptor in liver cells, inhibiting the growth of cancer cells. These studies have showed that the expression of oncogenes such as cyclin-dependent kinase 2 (CDK2), cyclin E and phospho-Rb decreased in liver cells and also the expression of protein p21 increased then apoptosis induced [70]. Other studies have also reported that T3 reduces the expression of MMP2 through Dickkopf-related protein 4 (DKK4) which reduce the metastatic progression of cancer [70].

Conversely, T3 binding to THRs, may be involved in increasing the strength of liver cell metastasis. T3 makes this increase likely due to increased expression of lipocalin 2. Lipocalin 2 and THRα were both overexpressed in samples from HCC patients and were associated with cancer grade, stage and survival. In the other word, binding of T3 to THRs increased lipocalin in hepatocytes and by binding to integrin increased the growth and progression of liver tumors through ERK1/2 and Akt [20]. Integrin avb3 plays an important role in physiological and pathological processes such as bone resorption, wound healing, angiogenesis, tumor invasion and metastasis. Studies have also shown that overexpression of avß3 is associated with the development of cancerous tumors [71]. There is a significant correlation between the expression of OPN and avß3 in HCC. OPN has been shown to act through avß3, and activate PI3K/Akt signaling. Thus, overexpression of OPN and avß3 may have a synergistic effect in promoting hepatocarcinogenesis, jointly participating in proliferation, transformation and regulation of migration in liver cells [14].

CRC is the second leading cause of cancer death worldwide [3]. Many anti-cancer drugs have been introduced in the world to treat cancer, especially colon cancer. Surgical techniques for managing liver disease in CRC have helped improving the overall survival of CRC patients. But according to many researches and studies that have been done in connection with cancer treatment, the cure for colon cancer has not been significant. This rate remains below 15% and metastatic CRC (mCRC) is significantly incurable [4].

It has also been shown that thyroid hormone increases the nuclear accumulation of High-mobility group AT-hook 2 (HMGA2) and β-catenin in a concentration-dependent manner in colorectal cancer cells with different k-RAS statuses. The β-catenin pathway plays a key role in signaling in the evolutionary process, but also in the development of several malignant neoplasms, including colon cancer [72]. In a study by Vonlaufen et al., 2001 reported that the lower the expression of αvβ3 in patients increased the survival of patients with colorectal cancer. One study had shown that positive expression of αvβ3 is seen in patients with colon cancer [73]. Also a study of 20,990 people with colon cancer found that people with long-term hypothyroidism reduced their risk of developing colorectal cancer. Therefore, it seems that low levels of thyroid hormones provide conditions that reduce the function of their receptors, so the process of proliferation and growth of cancer cells is reduced [12].

Several study showed that αvβ3 expression levels were closely correlated with patient survival data and tumor metastatic potential. Patients who are in more unfavorable conditions have expressed higher levels of αvβ3. This increase in αvβ3 expression is seen not only in small vessels but also in large vessels. Determination of αvβ3 receptor levels seems to be used as a prognostic factor in colon cancer [12].

In colon cells, by binding αvβ3 receptor to its ligand, EGFR is dimerized and autophosphorylated, and the tyrosine kinase EGFR is activated and generated the signal that the cell proliferation initiated. Any factor that activates EGFR, inactivated apoptosis and activated the proliferation of the cell. THs are molecules that are involved in the activation of EGFR and therefore induced the proliferation of cancer cells. Of course any defect in thyroid hormone receptors causes a defect in the development of the affected organ. The relationship between thyroid hormones (T4 , T3 ) and αvβ3 is important in the process of colon cell proliferation because the EGFR factor is one of the most important factors in the process of inducing cell proliferation [75].

This review showed that in liver and colon cancer cells, when thyroid hormones bind to the avβ3 receptor, also the pathways mechanism in these two cancers are similar. Although there has been controversy regarding the effect of thyroid hormones and liver cancer, the role of avβ3 integrin in cancer progression has been clear. Several studies showed that αvβ3 expression levels were closely correlated with patient survival. Patients who are in more unfavorable conditions have expressed higher levels of αvβ3. Our study showed that the expression of avβ3 is associated with tumors progression in liver and colorectal cancer. Increased integrin expression plays a vital role in angiogenesis, which inhibits apoptosis and stimulates cells to become cancerous. Also, the high expression of αvβ3 integrin on blood vessels in tumor tissue and tumor cells makes αvβ3 integrin a key target for anticancer drugs. Thus, αvβ3 plays an important role in the diagnosis and prognosis of colorectal and liver cancer. Determination of αvβ3 receptor levels seems to be used as a prognostic factor in colon and liver cancer.

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Conflict of interest: The authors declare no conflict of interest.

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