الفهرس 
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Abstract
Breast cancer is the most common cancer in women worldwide, and the second most common cause of death from cancer overall. Increased incidence of cancer in recent years and its impact on different physical, mental, and social dimensions of human life have turned it to a major problem of the century. This disease is the primary cause of mortality among women aged 45–55 years.The incidence of breast cancer is almost 1-in-8 women, requiring complete tissue removal, chemotherapy, radiotherapy, and hormone therapy most of the time. The incidence and mortality rates of breast cancer represented 24% and 15% respectively of all cancer types among females in 2018.
BC may be attributed to many risk factors, that have a great influence on the development of the malignant tumor, including modifiable risk factors such as life style (obesity, alcohol, smoking, physical activity), radiation exposure, breast feeding, hormonal factor, and many other non-modifiable risk factors such as genetic factor (BRCA1, 2, tumor suppressor protein (TP53), Phosphatase and tensin), age and family history of breast cancer.
The Epithelial-Mesenchymal transition (EMT) is a complex program in which epithelial cells acquire a mesenchymal phenotype and motility through a cascade of biological events. It is a natural phenomenon in development and wound repair but also represents one of the “hallmarks of cancer”. During tumorigenesis, changes in EMT regulatory pathways lead to a loss of cellular adhesions, changes in the polarization of the cell and cytoskeleton, detachment, migration, intra-vasation, and survival in the vascular system; extravasation, and finally, metastasis.
Calcium is a ubiquitous second messenger that plays a fundamental role in cellular migration, cell cycle control and apoptosis. A role for calcium channels in tumor proliferation has been reported in cell lines and tumors such as breast, brain, colorectal, gastric, hepatic, prostate, leukemia, retinoblastoma and pheochromocytoma, and silencing of calcium channel expression has been shown to reduce proliferation. Among the different calcium channels implicated in cancer, T-type voltage calcium channels (VGCCs) have been linked to tumor initiation and progression.
So the present study aimed to investigatethe possible role of(VGCC) in EMT and metastasis among breast cancer patients.
The patients were divided into the following two groups:
group I: 25 Non metastatic breast cancer patients
group II: 25 metastatic breast cancer patients
Sampling
1- Three (ml) blood was taken from all the contributors in the study
2- from each patient in group I, malignant breast tissue biopsy and corresponding macroscopically normal breast tissues from the same patient were taken.
The results of present study revealed that:
1- EMT genes PCR results
• E- cadherin in metastatic patients showed (5.2) fold increase when compared to non-metastatic patients in blood (P = 0.022*). On the contrary, its expression in tissue was significantly down-regulated in malignant tissue when compared to the counterpart TAN (0.042*).
• N- cadherin gene expression it was up-regulated at the level of both blood and tissue where in metastatic compared with non-metastatic patient there was (3.3) fold increase with (P = 0.038*) in blood, while in tissue there was a high significant increase in malignant tissue compared to their TAN counterpart (P = 0.015*).
• Snail gene expression was increased in metastatic when compared with non-metastatic patients in blood (P = 0.045*), beside (5.44) fold increase. Malignant tissue showed difference in its expression compared to TAN (P = 0.568).
• Vimentin gene expression was up-regulated by (1.23) fold increase and (P = 0.028*) in blood of metastatic compared to non-metastatic patients, besides it was significantly elevated in malignant tissue compared to their TAN (P = 0.021*).
2- TTCC genes PCR results
• CACNA1G was significantly decreased in blood of metastatic patients compared to non-metastatic patients with (0.7) fold decrease and (P = 0.044*). It also showed a highly significant decrease in malignant tissue compared to TAN (P = <0.001*).
• Both CACNA1H, CACNA1I showed the same results, where they were significantly increased in metastatic compared to non-metastatic patients in blood (P = 0.002* and P = <0.001*) respectively, beside a high fold increase of (3875) and (2219) increase.
• Malignant tissue showed significantly elevated expression of CACNA1H compared to its TAN (P = 0.04*). On the other hand no significant difference was found for CACNA1I gene (P = 0.549).
3- Correlation studies
• E- cadherin expression showed negative but not yet significant correlation with vimentin as mesenchymal markers in blood of non-metastatic (r = - 0.468, P = 0.058). Tissue also showed strong positive correlation between N- cadherin and vimentin (r = 0.629, P= 0.012*), the strongest correlation was found between the mesenchymal markers N- cadherin and Snail in blood of metastatic patients (r = 0.816, P = <0.001*) confirming their role in metastasis.
• To evalute the role of T-type VGCC in EMT, correlation studies were preformed. In blood of non-metastatic patients correlation between T-type VGCC and EMT genes revealed positively and negativly but not yet significant correlation. CACNA1G was positively correlated with E-cadherin (r = 0.488, P =0.055). In tissue of non-metastatic BC patients positive correlation was found between CACNA1H and each of N- cadherin (r = 0.643, P = 0.010*), vimentin (r = 0.647, P = 0.005*). In addition CACNA1I showed
strong significant positive correlation with N- cadherin (r = 0.819, P = <0.001*), vimentin (r = 0.588, P = 0.005*) and CACNA1H (r = 0.484, P = 0.031*). In blood of metastatic BC patients, there was a strong positive correlation between CACNA1H and N- cadherin (r = 0.549, P = 0.010*) and Snail (r = 0.491, P = 0.024*).
• In an attempt to evaluate the relation between blood and tissue expression level for each gene, correlation studies were performed. No significant correlation was found (P >0.05).
from the present study we could conclude the following, although EMT associated genes were differentially expressed between breast cancer tissue and their adjacent normal tissue, as well as in blood of metastatic and non-metastatic breast cancer patients,collectively there was a tendency for the mesenchymal genes to be up-regulated or highly expressed in blood of metastatic patients compared to non-metastatic as well as in malignant tissue compared to their adjacent normal. This clarifies and confirms the role of EMT in metastasis.
The presence of strong correlations between the expression of Ca2+ T-type genes and mesenchymal genes may reflect their role in EMT through the possible up regulation of both N-cadherin and vimentin leading to the loss of cellular adhesion, change in polarization of cells, migration and finally metastasis.
Recommendation
1. Further studies should be performed on larger sample size to obtain more reliable results that could reflect the possible role of T-type VGCC in EMT .
2. Stratification of the different histological and molecular subtypes of breast cancer should be taken in consideration to avoid the great variationforthe same gene expression among patients.
3. To achieve more verification for the role of T-type VGCC in EMT, proteins and transcription factors involved in Ca2+ signaling should be extensively studied.
4. Lately, in our researches preformed on BC, most of the patients at presentation were at late stage, so these should be more awareness ofthe regular check-up for females above age of 40.
5. Calcium channels blockers usually prescribed for cardiovascular and hypertensive patients, some of them act by anti-apoptotic mechanisms, therefore such drugs should be prescribed with caution forpeople at increased cancerrisk .