Impact of Vitamin D on Expression of SIRT7 and CYP24A1 in Human Breast Cancer Cells

INTRODUCTION

Breast cancer (BC) remains primary cancer in women worldwide, both in developed and developing countries.^[1,2] Despite the recent treatment approaches, including surgery, endocrine therapy, and targeted therapy, the total number of people diagnosed with cancer has nearly doubled in the past two decades.^[3] Therefore, a deeper understanding of the BC pathogenesis process in molecular signaling pathways is of crucial importance.^[4] Sirtuin seven (SIRT7) is one of the seven members of the sirtuin family (SIRT1–7) and belongs to Class III histone protein deacetylase (HDAC), a type of oxidized nicotinamide adenine dinucleotide (NAD +)-dependent deacetylase.^[5] Sirtuins were first discovered in yeast to change replication time.^[6] SIRT7 is found primarily in the nucleus with histone deacetylase activity and non-histone proteins.^[7] SIRT7 has different physiological functions such as neurogenesis and cell survival, genomic stability by interacting between various proteins and is believed to have an essential role in the regulation of DNA damage repair.^[8] Recent studies have shown that SIRT7 can modulate the activation of RNA polymerase I and III and influence the transcription of ribosomal RNA and Polymerase II, as well as the biosynthesis of proteins such as GABPβ1,^[9] and the acetylation activity of p53.^[10] Cytochrome P450 subfamilies Family 24, Part 1 (CYP24A1) is found in the inner part of the mitochondrial layer of cytochrome P450 and encodes hydroxylase 24, the protein necessary to inactivate the dynamic form of Vitamin D (1, 25-dihydroxyvitamin D3) (Calcitriol) and controls the synthesis of Vitamin D.^[11,12] The main function of Vitamin D is to regulate osteosynthesis and calcium homeostasis.^[13] Dietary Vitamin D converts to calcitriol which is a steroid hormone in the liver.^[12] Different studies have been conducted to identify the role of Vitamin D in cancer prevention, based on its relationship with different prognostic factors such as tumor stage, degree, size, and lymph node involvement.^[14,15] Recently, an inverse relationship between Vitamin D concentration in plasma and cancer incidents has been discovered.^[16] Calcitriol can modify genes expression and different signaling pathways.^[17] A recent discovery indicates the regulatory role of calcitriol on different genes expression and microRNAs expressions.^[17] The meta-analyses study has shown that a high level of circulating 25 (OH) D can reduce BC incidence in women.^[18]

Calcitriol can influence cell differentiation and proliferation by regulating the expression of many factors in tissue physiological conditions.^[19] Studies have established the connection between the concentration of Vitamin D and carcinogenesis, in vivo and in vitro.^[14,16,17]

CYP24A1 is an oncogene in human cancer and affects the level of expression of several signaling molecules related to carcinogenesis. Different studies have elucidated the important effect of CYP24A1 in the dysregulation of cell growth by reducing calcitriol concentrations. The loss of function mutations in CYP24A1 leads to increased levels of active Vitamin D metabolites.^[19-21] Therefore, discovering how Vitamin D affects cancer initiation will help to develop efficient therapeutic strategies in BC.^[22] In this work, we investigate the expression levels of CYP24A1, SIRT7, and their clinical connection in BC tissue. After Vitamin D treatment, we also examined the expression level of this gene in BC cell lines (MCF7 and MDAMB231) to understand the impact of Vitamin D on the expression of the CYP24A1 and SIRT7 genes.

MATERIAL AND METHODS

RESULTS

Detection of CYP24A1 and SIRT7, mRNA, and in tissues and cell lines

To define the role of Vitamin D (1,25-dihydroxyvitamin D3) on the expression of CYP24A1 and SIRT7, we measured CYP24A1, SIRT7, and expression levels using qRT-PCR.

CYP24A1 and SIRT7 were both measured in tissues and cell lines. CYP24A1 and SIRT7 expression was significantly upregulated in BC tissues compared with paired normal adjacent tissues (P < 0.01) [Figure 1], and in cell lines [Figure 2], compared to normal cell lines MCF-10A before and after the Vitamin D treatment. The results showed Vitamin D stimulates CYP24A1 and SIRT7 expression in BC.

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Figure 1:

(a and b) The relative expression level (ΔCt) of CYP24A1 and SIRT7 in breast cancer (BC) samples compared with controls. The statistics indicate that these genes were upregulated in BC samples compared with controls (P < 0.01)

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Figure 2:

Vitamin D (1,25-dihydroxyvitamin D3) affects the expression of CYP24A1 and SIRT7. Expression profile of CYP24A1 and SIRT7 in BC cell lines (MC7- MDA-MB-231) before Vitamin D treatment (P < 0.05) (a) and after Vitamin D treatment (P < 0.0001) (b).

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The potential diagnostic values of CYP24A1 and SIRT7 in BC

We analyzed the potential diagnostic values of CYP24A1 and SIRT7 in BC. ROC curve analysis was conducted to predict the potential of CYP24A1 and SIRT7 expression as a novel diagnostic or prognostic bio markers between tumor tissues and control [Figure 3]. The area under the ROC curve for CYP24A1 and SIRT7 was 0.77 and 0.84, respectively, and the sensitivity and specificity of CYP24A1 and SIRT7 were 0.75 and 0.63 for CYP24A1; 0.83 and 0.73 for SIRT7.

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Figure 3:

Potential diagnostic values of CYP24A1 and SIRT7 in breast cancer. AUCs of CYP24A1 and SIRT7 were 0.77 and 0.84, respectively. With the sensitivity of 0.75 and specificity of 0.63 for CYP24A1 and 0.83 and 0.73 for SIRT7, respectively.

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DISCUSSION

BC is still the most common malignancy and the first main cause of death in women, unfortunately, most patients have no specific symptoms in the early stages of BC and are diagnosed at advanced stages. Therefore, early diagnosis of the disease is essential for better treatment.^[2] SIRT7 belongs to the histone deacetylase family, which can interact with different signaling pathways.^[24] The role of sirtuins 7 has been reported in various pathogenesis and diseases, including cardiovascular disease, Type II diabetes, cancer, and neurodegenerative diseases.^[8,9] SIRT7 expression is upregulated in metabolically active cells, and this upregulation of SIRT7 may contribute to the malignant tumor phenotype and lead to tumor genesis.^[25] SIRT7 is believed to play an important role in tumor initiation, tumor progression, and the promotion of cell growth.^[9] Its monogenic role has been studied in other malignancies such as human ovarian, liver, bladder, and colon cancer.^[10] Mitochondrial inner membrane cytochrome enzyme CYP24A1 was nominated as an oncogene in the development of several cancers.^[12] Studies have reported two Vitamin D-responsive elements (VDREs) at the upstream of the CYP24A1 gene, these VDRE sites can regulate CYP24A1 expression synergistically and play a critical role in the metabolism of Vitamin D (calcitriol) performs a different biological function, such as inducing apoptosis, modifying calcium and phosphate homeostasis, regulating hormone-dependent genes, and inhibiting proliferation.^[26] Studies showed that the inhibitory role of calcitriol on cell proliferation might change to a vital role depending on the cell differentiation degree and concentration of Vitamin D. [27,28] Although the inverse association of Vitamin D and BC is studied in many meta-analyses, still there is an unexpected result about the higher concentration of 25(OH)D and BC risk.^[13,29] Vitamin D also can increase the expression levels of different growth factors such as keratinocyte (KGF) and increase the proliferation ability of cancer cells.^[14] Previously, we showed that the treatment of Vitamin D can modulator the expression of SIRT1 in BC cell lines (MCF-7 and MDAMB-231).^[23,30] Another study has shown the role of an active form of Vitamin D in the progression of many metabolic-related diseases and showed that Vitamin D treatment can improve SIRT1 levels in serum levels of patients with Type 2 diabetes.^[31] Since the significant role of calcitriol on the regulation of different genes is not fully understood, in this research, we aimed to evaluate the impact of Vitamin D on the expression of SIRT7 and CYP24A1 an enzyme related to activation of Vitamin D which both were upregulated in different tumor tissues. We also explored the correlation between these selected genes and clinic pathological characteristics in BC patients and found that the expression of CYP24A1 was significantly correlated with tumor stages (P = 0.03), and both CYP24A1 and SIRTS showed a potential to be assessed as the diagnostic biomarker in BC based on their ROC curves (0.77 and 0.84, respectively). We also determined the role of Vitamin D treatment on the regulation of CYP24A1 and SIRT7 expression in BC cell lines. Data showed Vitamin D treatments significantly induced expression of CYP24A1 and SIRT7 in both cells. In conclusion, upregulation of CYP24A1 and SIRT7 expression in tissues may play essential roles in BC progression and has the potential to be used as a diagnostic biomarker in distinguishing BC tissues from normal tissues. It may be concluded that Vitamin D treatment may modulate gene expression profile in a cell-specific manner in BC.