Increased levels of cellular iron is vital for growth and rapid proliferation of the cancer cells. Various studies portrayed the importance of dysregulation of proteins enrolled in iron metabolism, have vital parts in malignant transformation. Alterations of genes related with iron hemostatsis had been thought to be prognostic biomarkers and therapeutic targets in malignancies. Miller et al.’s study reported that attrition of genes related with iron excrition and in addition to this the upregulation of iron uptake process had effects on the prognosis of breast cancer [12].
The TFRC gene encodes TfR1, a receptor located in cell surface, esential for the iron absorption into the cell. TfR1 had increased expression in cells with rapid proliferation, like cancer cells, which have high demang of iron during the proliferaiton. Based on this knowledge, the upregulation of the TFRC gene had been defined as a target candidate in cancer therapy. Increased expression of this gene was found to be associtaed with cancer cell proliferation and poor survival in patients with breast malignancy, in a report by Habashy et al [13]. Jiang et al. reported the expression of TFRC was elevated in malignant breast cells (MCF-7 cells) than cells in the normal mammary glands(MCF-12A cells) [14]. In allignment with these prior findings, in our study the expression levels of TFTC was significantly upregulated in the malignant breast cells.
Transcriptional, post-transcriptional processes effect the TFRC expression. The iron responsive element system (IRP1/2-IRE), controls the iron homeostasis through post-transcriptional modulation of the TFRC through a sophisticated system. Chen et al. showed that deprevation of iron led to the binding of IRP1/2 to IREs, which led to the stabilization of the mRNA of TFRC, thus raising the expression of proteins [15]. Wang et al. showed that induced IRP1 expression in malignant breast cells (MCF7) resulted in elevation of mRNA and protein levels of TFRC [16]. The expression level of IRP1 gene was reduced in the malignant cells in our study. Therefore, we can not postulate the relationship among the elevation of the expression of TFRC and expression levels of IRP1 gene. Wang et al. showed that overexpression of IRP2, was associated with reduced ferritin heavy chain and TfR1 elevation. However this relationship was not present for IRP1. Wang et al, reported that IRP2 expression alone was associated with the molecular subtype and the histological grade of the human breast cancer [16]. Increased levels of TfR1 expression were reported in prostate cancer cells [17]. In a study by Deng et al., the expression level of IRP2 was detected in 4 malignant prostate cell lines, IRP1 expression level had moderate risein only 2 cell lines [17]. The increase in the regulation of IRP2 in malignant prostate cells homogenizes normal iron control pathways, which gives way to elevation in the levels of iron, aiding tumor proliferation. IRP1 was reported to be downregulation in hepatocellular carcinoma and this could be used to predict the stage of the tumor and the prognosis [18]. Oxygen, oxidative stress and the levels of nitric oxide are among the additional molecules and processes associated with the respond of IRPs in addition to cellular iron levels. It was reported by Luo et al. that hypoxia in the short term lead to a decline in mRNA of IRP1and levels of protein in HepG2 cells [19]
The most essential iron efflux transporter is ferroportin protein (FPN1). Its function is the siphoneage of the iron to the cytoplasm of the macrophages and the enterocytes. FPN1 is encoded by the SLC40A1 gene. Its expression if controlled by hepcidin, encoded by the HAMP gene [20]. The dysregulated hepcidin–FPN1 signaling was related with rise in malignant transformation by experimental and epidemiological studies.. However, the exact processes associated with the dysregulation of iron-related gene expression in the malignant processes are still not uncovered. In our study, no difference in the hepcidin gene expression was identified. On the other hand, a statistically insignificant decrease in the FPN1 gene expression was seen. The rise in hepcidin leves were seen in the lung, breast, prostate, renal cancer in addition to various other cancers. However, its levels were reduced in some brain tumors and hepatocellular carcinoma [21]. Shen et al. reported the findings in hepatocellular carcinoma, where the levels of hepcidin was reduced in malignant cells than the normal cells [22]. In breast cancer, Zhang et al. reported limited elevations in the expression levels of hepcidin in malignant cells than the normal tissues [23]. Similar to our study, the tumor ferroportin concentration was found to be low in the breast tumors in that study. In addion, Pinnix et al.'s study revealed that the mRNA level of tumor hepcidin was able to discern only marginal significance for patient prognosis [24]. The major and most prominent hepcidin expression simulators are bone morphogenetic proteins (BMPs).. Reports have shown that BMP6 was not alone, other BMPs also regulate hepcidin expression [21]. The UniGene database’s in silico analysis showed that, malignant breast tissue had reduced expression of ferroportin [25]. Our study showed that ferroportin gene expression in the cancer tissue was also found to be 0.627 times reduced compared to the normal tissue.
The coreceptor of BMP, hemojuvelin’s clevage blocks the BMP-SMAD signaling pathway, through inhibition of the expression of hepcidin by matriptase-2 [26]. In our study, matriptase-2 expression increased (1,912 fold) and hemojuvelin gene expression decreased (0.531 fold) in the malignant breast tissues. In our previous study that we conducted in breast cancer patients, the levels of the expression of TMPRSS6 gene in the malignant tissues were 1.88 fold elevated than normal tissues [27]. Normal breast tissue samples was reported to have dominant matriptase-2 expression [28]. Overall et al.’s study showed that the gene expression levels of TMPRSS6 was elevated in the invasive ductal carcinoma cells [29]. Gitlin-Domagalska et al. showed the relationship of TMPRSS6 with decline in gene expression levels, progression of tumor cell, and poor prognosis [30].
We could not find any literature investigating homojuvenile gene expression in breast cancer. The simulation of hepcidin transcription by BMP is inhibitted by matriptase-2. This process is achieved by the cell surface proteolytic processing of the BMP co-receptor hemojuvelin [28]. Controversial results had been reported on BMPs role in malignancies of the breast. The BMP6’s role in breast malignancies had been found to be variable according to several studies, performed on the relationship between hemojuvelin and BMP6. Most of the reports present that BMP6 expression is reduced in the malignant breast tissue [31]. Our study showed that the gene expression of TMPRSS6 elevated in the malignant tissues. An hypothesis could be constructed on the interaction between matriptase-2 with the BMP6/SMAD signaling via by hemojuvelin. The low homojuvenile expression we found in our study supported this hypothesis.
Hepatic miR-122 expression had paramount importance in sustaining the iron levels in the liver andthe plasma. Castoldi et al. reported that the inhibition of the expression of hepatic miR-122 in the liver tissue of mice and in primary murine hepatocytes, lead to a rise of mRNA expression of hepsidine and homojuvenile [10]. We portrayed that of miR-122 expression was reduced in the malignant breast tissues. On the other hand, in our study, we found a decrease in expression of homojuvenile in malignant breast tissues, while the hepcidin expression was unchanged. miR-122 was reported to be essential in the regulation of serum cholesterol levels [32]. Therefore, the overlapping or non-overlapping metabolic pathways in which miR-122 plays a role may alter the effects of mir-122 on gene expressions. On the other hand, various studies studied the role of mir-122 in malignant breast tissue. Similar to our study, Yan et al reported that miR-122 levels were reduced decreased in malignant breast cells than the normal tissue samples; and this finding was statistically significant [33]. Two studies portrayed the miR-122 downregulation in malignant breast cells [34, 35]. Several other studies showed that expression of miR-122 elevated in malignant breast tissue than the normal tissue [36].
In conclusion, our study shows a powerful associatin between genes enrolled in the metabolism of iron and the breast cancer, paving the way for the establishment of new tools to be used for the prognosis of breast cancer. They could also aid in the establishment to find new methods to identify normal and malignant breast cells, which could be utilized for to therapeutic advantage.