Comparing the expression of TTK, ARMC3 and TPTE genes in tissue samples of breast cancer patients with normal tissue

Master thesis in medical biotechnology

Abstract:

Introduction: Breast cancer is still the most common cancer among women. Biomarkers expressed in cancer tissue play an important role in diagnosis, prognosis and response to treatment. Cancer-testicular genes are often expressed in normal testicular tissue, but the expression of some of them has been reported in some types of cancer. Because the testis is a protected place from the immune system, if these genes are expressed in the tumor, they can be used as suitable targets for breast cancer immunotherapy.

Research method: after obtaining 95 samples including 40 tumor samples, 40 samples adjacent to the tumor and 15 normal samples from the biobank, RNA extraction was performed from them. Then, the extracted RNAs were DNase treated and cDNA was made from them and the expression of ARMC3, TPTE, TTK genes along with ACTB (internal control) was analyzed by Real-Time PCR method. Result: 43.6% of the tumor tissue and 25.6% of the tissue adjacent to the tumor expressed the ARMC3 transcript.  ARMC3 increased expression in 41% of tumor samples and decreased expression in 46.2%. Also, the expression of this gene was unchanged in 12.8% of tumor samples. These changes were statistically significant. 21.6% of the tumor tissue and 8.1% of the tissue adjacent to the tumor expressed TPTE transcript. TPTE increased expression in 34.1% of tumor samples and decreased expression in 58.5%. Also, the expression of this gene was unchanged in 7.3% of tumor samples. These changes were significant in all three groups. 50% of the tumor tissue and 20% of the tissue adjacent to the tumor expressed TTK transcript. TTK increased expression in 45% of tumor samples and decreased expression in 50%. Also, the expression of this gene was unchanged in 5% of the tumor samples, which were significant in the increased and decreased expression groups.  It should be noted that none of the normal samples expressed the investigated genes and all the samples expressed the ACTB gene.

Discussion: Since these genes had a higher expression in the tumor sample compared to the one adjacent to the tumor, and also because they had no expression in the normal sample, they are important. Also, the expression of some of these genes in the tissue adjacent to the tumor may be related to the cancerous stage, and it may be because these tissues are affected by the oncogenic and epigenetic changes of breast cancer. According to these points, these genes may be considered as candidates for suitable biomarkers for breast cancer.

Key words: breast cancer, cancer-testicular genes, biomarker, immunotherapy

Introduction

Breast cancer is the most common cancer in women (23% of all cancers ), with the occurrence of more than one million new cases per year worldwide, and it is still the main cause of cancer-related deaths in women with 411,000 annual deaths (14% of cancer-related deaths in women). The mortality rate of breast cancer in Iran is reported to be 1.7 per 100,000 cases, the age-standardized ratio of this disease is estimated to be about 24 cases per 100,000 women per year, and considering that Iran has about 35 million women, this statistic represents 7,000 new cases of breast cancer per year (3-5).

Since women It is the center of gravity of the family and with the involvement of the woman, all the members of the family and even the upbringing of the children will be affected. Today, breast cancer treatment is done through surgery followed by chemotherapy or radiation therapy or both. Breast cancer has a good prognosis in the early stages and is generally treated through lumpectomy and radiation therapy. More advanced cancers are generally treated through surgery (lumpectomy or mastectomy) and chemotherapy and sometimes radiation therapy. Metastatic breast cancer is almost incurable and is controlled through a combination of treatments (6, 7).Despite the increase in the prevalence of breast cancer, its mortality has decreased due to changes in treatment methods. Nevertheless, a significant percentage of all cancer cases and deaths related to it are related to breast cancer (8). Finding new cancer treatment methods seems very necessary and immunotherapy is considered as a revolution in cancer treatment today. One of these methods is the preparation of an anti-cancer vaccine, which is used to detect and destroy cancer cells by stimulating the immune response. Some vaccines can be based on specific antigens, such as CT antigens (cancer-testis antigens), which are specifically expressed in tumors and have limited expression in other tissues (9). CT antigens were first discovered in 1991 by Bruggen and his colleagues when the first tumor antigen significantly changed the immunological response of the tumor. Through cytotoxic T lymphocytes derived from tumor patients in the laboratory (in vitro) they identified the mz2 E gene that was expressed in human melanoma cell lines (10). They can be immunogenic and are regularly recognized by the immune system of cancer patients, so they can be suitable candidates for cancer immunotherapy and can be used as biomarkers for early cancer diagnosis (11). It was on this basis that Scanlan and colleagues in a study in 2002 introduced CTAGs as suitable targets for cancer immunotherapy (12). Testicular cancer genes express a heterogeneous group of immunogenic proteins (cancer antigens - testis Antigen = CTAG Cancer/Testis) which are often exclusively expressed in normal tissue. testis and percentage of different types of tumors are expressed. These antigens are proteins that are naturally expressed in male reproductive cells. Most of these antigens are expressed in the testes and some of them in the ovaries and trophoblasts. Premature reproductive cells (oogonia and primary oocytes) express these antigens, but their expression has not been seen in follicles and oocytes. In the testis, the presence of these antigens is usually in the interstitial space of the testis, and these cells are never seen in the seminiferous tubules. According to the information available in the Cancer/Testis Gene Database, more than 150 members of this cancer-testis gene family have been identified (13). Based on the tissue specificity and immunogenicity of Cancer Testis Antigens and because they are not expressed in most normal tissues, therefore, if they are expressed in tumor tissue, they can be used as suitable candidates for cancer immunotherapy and the preparation of cancer vaccines based on these antigens. It should be noted that different types of these genes are expressed in different samples of tumoral tissues, and despite the many commonalities in their expression, their quantity is different in each tissue. Based on this, the expression quantity of these genes is also important in the immunotherapy of cancers (14). Among the positive points of using immunotherapy methods in the treatment of cancer and such vaccines, we can mention the few side effects of these methods. Many phase 1 studies in which different types of these vaccines have been used for a number of malignancies have pointed to the harmlessness of these methods (15). Biomarkers that express in cancer tissues have a significant role in the diagnosis, prognosis and response to treatment. Cancer-Testis Genes are expressed predominantly in the testes, but the expression of some of these genes in different cancers have also been reported. Because the testis is protected location from immune system, if these genes are expressed in cancerous tissue, can be used as targets for cancer-specific immunotherapy