1.1. Breast cancer Cancer („Karkinoma‟ in Greek and in Latin for crab) is a enigmatic heterogeneity disease caused due to association of genetic, epigenetic and environmental factors, which arises from a clone of cells that amplify in an uncontrolled manner because of somatically acquired genomic defects (Bertucci & Birnbaum, 2008; Creighton et al., 2010; Downs-Holmes & Silverman, 2011). Breast cancer („cancer mamae‟) is a type of cancer that either originates in the breast tissues or is primarily present in the breast cells (Sariego et al., 2010). Breast cancer, is the most frequently occurring lethal neoplasm in women globally and it is leading in both incidence and mortality of women in developing countries (Jemal et al., 2010). Breast cancer is the most common form of “malignant neoplasm of the breast” and the second leading cause of cancer mortality among women. 1.2. Epidemiology of breast cancer Breast cancer is one of the most common invasive cancers in women with greater than 1,300,000 new cases and 450,000 deaths were reported each year globally (Kamangar et al., 2006; Koboldt et al., 2012). Breast cancer is the second most common form of cancer amongst females and also the fifth most cause of cancer deaths globally. Each year more than 1.3 million women are being diagnosed with breast cancer worldwide and approximately 4.52 million die due to this disease (Bleyer et al., 2012). The rate of occurrence of breast cancers in females vary geographically, with the highest frequency of 1 in 4 female cancers in North America and Western Europe (Bray et al., 2004), to a low frequency of 1 in 16 female cancers in China and Japan (Patlak et al., 2001). In the United States alone, one out of every eight women was diagnosed with breast cancer if all live to their full life span (Downs-Holmes & Silverman, 2011). In India, non-communicable diseases including cancer are emerging as major public health problems (Itagappa et al., 2013). Cancer has become one of the ten primary causes of deaths in India (Gupta et al., 2003). National Cancer Registry (NCR)-based on the cancer registry data forecasts that there would be about 800,000 new cancers cases in India every year (Parihar et al., 2012). Over 7 Lakh new cases and 3 Lakh deaths occur annually due 1
to cancer (Rao et al., 2003). The incidence rate varies between urban and rural women; the incidence in Mumbai is about 27 new cases per 100,000 women per year while in rural Maharashtra it is only 8 per 100,000 (Chopra, 2001). These discouraging numbers, reflecting the increasing rates of incidence and death are related to the lack of improvement in detection and diagnostic methods and the paucity of breakthroughs in treatment regimens. Breast cancer is the most common non-skin cancer in women and the second most common cause of cancer-related death in women, which account for almost 20% of all malignancies. Over half a million women develop breast cancer every year. Worldwide, it is estimated that 1.4 million women are diagnosed with the disease per year, which accounts for about 23% of all cancers. Around 5,00,000 breast cancer related deaths are reported annually (American Cancer Society, 2011). According to National Cancer Registry Programme (NCRP), the number of breast cancer deaths in India would increase to 106,124 in 2015 and to 123,634 in 2020 (Takiar et al., 2010). 1.3. Types of breast cancer Breast cancer classification is based on growth, size and stage of the tumor. Each breast has 15 to 20 sections called lobes, which are made up of many smaller sections called lobules. Lobules have groups of tiny glands that can make milk. The lobes and lobules are connected by thin tubes, called ducts (Figure 1.1). Breast cancer is a tumor that starts from cells of the breast tissue, either in cells that line the ducts that carry milk to the nipples called ductal cancer. The ductal cancer is the most common type of breast cancer and about 7 of every 10 women with breast cancer have ductal carcinoma, which constitute about 80% of all breast cancers. Nearly, all women with cancer at this stage can be cured1 . Cancer that starts in lobes or lobules is called lobular cancer. It is more frequently occurring in both breasts than other types of breast cancer. About 1 of every 10 women with breast cancer has lobular carcinoma. Breast cancers are classified as non-invasive (in situ) and invasive (infiltrating). The term in situ refers to cancer that has not spread to other organelles f
of the body. These cancer cells cannot invade into the surrounding breast tissues. Invasive breast cancer has a tendency to spread (metastasis) to other tissues of the breast and/or other regions of the body such as bones, lungs, distant lymph nodes, skin, liver and brain. Each type can be either invasive (cancers that spread to other parts of the body) or in situ (one that does not). A less common types of breast cancer include inflammatory, medullary, mucinous, papillary, tubular and Paget's disease (WHO, 1981; Engel, 1996).
Figure 1.1. Female normal breast anatomy2 Breast tumors can be benign or malignant, the former are not life-threatening, but the malignant breast tumors are cancerous which can invade the surrounding tissues or metastasize to other parts of the body through the lymphatic system (lymphatic vessels and lymph nodes), such as the liver and bone. If cancer cells have spread to the surrounding lymph nodes, the tumor will enter the bloodstream and metastasize to other parts of the body (Schottenfield & Joseph, 1996). Breast cancer occurs mostly in women but a small population of men is also affected by it (Kelsey et al., 1990; Wu et al., 2002). Breast cancer continues to be a most important burden that cause of death among women worldwide (Greenlee et al., 2000).
1.4. Stages of breast cancer The stage of breast cancer depends on the size of the breast tumor (Figure 1.2) and whether it had spread to lymph nodes or other parts of the body. The TNM classification for staging of breast cancer is based on the size of the cancer when it originally started in the body and then the locations to which it has travelled. Presently, the staging systems in classification of breast cancer is based on the clinical size and degree of invasion of the primary tumor (T), the clinical absence or presence of palpable axillary lymph nodes and confirmation of their local invasion (N), besides the clinical and imaging evidence of distant metastases (M). TNM classification which has been subdivided into Stage-0 called carcinoma in situ (lobular carcinoma in situ-LCIS) and ductal carcinoma in situ (DCIS) and the following four categories are classified by the Union Internationale Centre Cancer (UICC) (Fisher et al., 1987; Singletary et al., 2006): Stage I – During the early stage of breast cancer the tumor is less than 2 centimeter across but can not spread beyond the breast. The tumor is low-grade (likely to grow and spread slowly). Stage II - The tumor is larger than 2 centimeter but lesser than 5 centimeter but can not spread to the axillary lymph nodes. Stage III - Localised advanced breast cancer where the tumor is greater than 5 centimeter across and can spread to the lymph nodes under the arm; or the cancer is extensive in the underarm lymph nodes; or the cancer can spread to lymph nodes near the breastbone or to other tissues near the breast. Cancer could to 1 to 3 axillary lymph nodes or to the lymph nodes near the breastbone3 (found during a sentinel lymph node biopsy). Stage IV – Metastatic breast cancer can spread beyond the breast and nearby lymph nodes to other organs of the body, most often the bones, lungs, distant lymph nodes, skin, liver and brain4.
Source: Terese Winslow. U.S. Govt has certain rights (2007) Figure 1.2. The images of different stages of breast cancer 1.5. Risk factors Oncologists have identified a large number of risk factors for breast cancer such as: late age at first pregnancy (above 30 years), single child, menarche at a relatively early age (12 or younger) and, late age at menopause (Benz & Yuc, 2008), hereditary factors, carcinogenic elements, weight, race, tobacco (Itagappa et al., 2013), alcohol consumption (Holmes & Willet, 2004), obesity (Brown & Allen, 2002), shorter duration of breast-feeding (Katsouyanni et al., 1996), even night shift work etc., are some of the reasons reportedly causing breast cancer or the increased risk. A high fat diet is also identified as a risk factor. High fat diets during the pubertal age and obesity in the post menopausal age are risk factors for breast cancer (Rock & Demark-wahnefried, 2002). Lack physical activities are found to be protective for breast cancer (Bernstein et al., 1994). 1.6. Genetics of breast cancer Past several decades of cancer research has achieved an abundant and complex body of knowledge, revealing cancer to be a disease involving dynamic changes in the human genome. Somatic mutations that confer a growth advantage could be classified in to two categories: oncogenes and tumour suppressor genes. Oncogenic mutations are dominantly acting gains of function whereas tumour suppressor mutations are recessive losses of function. Previous studies on cancer research showed strong evidence suggesting that tumorigenesis in humans is a multistep processes which reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives (Hanahan & Weinberg, 2000; Medina & Rivera, 2010).
Mutation in BRCA-1 and BRCA-2 genes account for 5 to 10 % of breast cancer and are inherited through family history and these genes confer a high penetrance of disease risk (Miki et al., 1994; Wooster et al., 1995). In breast cancer some other genes such as CHEK2, BRIP1, ataxia telangiectasia mutated (ATM) and PLAB2 confer a 2-4 folds relative risk of breast cancer and they are classified as intermediate penetrance alleles (Ripperger et al., 2009). These mutations probably contribute to the large extent to early cancer development, though further mutation remains a controversial issue (Sieber et al., 2002; Skoulidis et al., 2010). Li Fraumeni syndrome is carrying mutations in Tumor Protein p53 (TP53) and Cowden disease is carrying mutations in phosphatise and tensin homologue (PTEN), and these have an increased risk of developing breast cancer, as well as other cancers (Lang et al., 2004; Rosemary et al., 2007; Liaw et al., 1997). Several other DNA-repair related syndromes involving genes such as: Serine/Threonine Kinase 11(STK11), PTEN, cadherin-1(CDH1), Neurofibromatosis Type I (NF1) and Nibrin (NBN) also increase the risk of breast cancer, but due to the rarity of these syndromes, their overall contribution to the population burden of breast cancer is small (Mavaddat et al., 2010). Every cell undergoes >20,000 DNA damaging events (Lindahl & Wood, 1999; Lange et al., 2011) and >10,000 replication errors per cell per day have been reported (Loeb, 2011). Therefore, mutations occur throughout the genome, and genes are responsible for maintaining genomic stability. Cancer-driving mutations occur mainly in a set of key genes that control cell proliferation (Johannes et al., 2012), differentiation as well as tissue and organ development (Stratton, 2011). A normal cell undergoes different essential physiological changes, to become a cancer cell sustaining proliferative signaling, escaping growth suppressors, resisting cell death (apoptosis), supporting replicative immortality, inducing angiogenesis (Pralhad et al., 2003) and activating invasion and metastasis (Meindl et al., 2011). Based on the number and pattern of recurrent mutations, over 400 genes have been identified as cancer genes (Futreal et al., 2004). Mutations in these genes cause deficiency in DNA repair and which lead to higher mutation rate in other genes (Friedberg, 2003). Another intensely studied aspect is that of germline mutations in BRCA-1 and BRCA-2 which are associated with chromosomal instability (CIN) in 6
~25% of hereditary breast cancer and 12% in ovarian cancer (Meindl et al., 2011). Clonal chromosomal abnormalities are frequently acquired during carcinogenesis due to the amplification, deletions, inversions and translocations (Mitelman et al., 2007; Heim & Mitelman, 2009). Chromosomal abnormalities lead to carcinogenesis through the various mechanisms viz: transcriptional deregulation of protooncogenes, duplication of proto-oncogenes, deletion or interruption of tumour suppressor genes. Oncogenic mutations typically alter the amino acid sequence of a protein, causing constitutive or inappropriate activation. For instance, point mutations are commonly observed in the RAS family of proto-oncogenes. KRAS mutations are common in lung, pancreas and colon carcinomas whereas NRAS mutations are often found in hematological malignancies like acute myeloid leukemia. The majority of mutations in these genes are in codon-12 and cause constitutive activation of the signal-transduction function of the RAS protein (Bos, 1989). 1.7. RAS is an oncoprotein RAS proteins (HRAS, KRAS and NRAS) are small (~21,000 Da) membrane-bound guanine-nucleotide binding proteins which are regulated by a GDP/GTP cycle, being inactive when bound to GDP (RAS GDP). RAS acts as an on/off switch that transduces extracellular signals (e.g. activated growth factor receptors)
Approximately 30% of all human malignancies contain an oncogenic point mutation in codons-12, 13 or 61 of one of the RAS genes (Shirahama et al., 2001). These mutations interfere with the intrinsic GTPase activity, rendering RAS constitutively active (RAS GTP) and leads to growth factor-independent signaling. Activating point mutations in the three human RAS oncogenes have been detected in a wide variety of tumors. The frequency of mutations and the RAS isoform that is predominantly mutated displays tissue specificity. KRAS is by far the most frequently mutated isoform (~80%), followed by NRAS (~20%) and HRAS (A transition and G>T transversion. In vitro and in vivo studies reported that KRASV12 regulated genes involve cytokine signaling, cell adhesion, cell survival, proliferation, apoptosis and colon development (Downward et al., 2003; Malumbres et al., 2003; Roberts et al., 2006; Haigis et al., 2008). KRAS mutations are responsible for the initiation of tumorigenesis. It is confirmed that signaling by activated RAS oncogenes also provide to the metastatic phenotype of tumor cells (Sanchez-Munoz et al., 2010). In order to progress to a metastatic carcinoma different genetic mutations have to accumulate. The point mutational (codon-12) activation of the KRAS oncogene may be a late event and could play a role in the metastatic progression of human breast cancer (Koffa et al., 1994). The KRAS protein plays an important role in tumor development, regulating downstream proteins that are involved in cellular process (Figure 1.4).
Figure 1.3. Model of the KRAS protein with G-domain highlighted (Jancik et al., 2010).
Figure 1.4. KRAS roles in breast cancer cell
1.9.Diagnosis of breast cancer 1.9.1. The past and future of breast cancer diagnosis Diagnosis of breast cancer can be carried out by the following diagnostic tools: 1. Mammogram 2. Breast ultrasound 3. Fine needle aspiration (FNA) 4. Ultrasound and FNA of the lymph Nodes 5. Biopsy 6. Magnetic resonance imaging (MRI) scan 7. Computerised tomography (CT) scan Observation of changes in breast tissue or the nipple during a breast examination or routine mammogram are often the first indication of breast cancer development. Imaging studies such as ultrasound, X-rays, MRI, or CT scans are used as diagnostic tests to assess the extent of disease progression (Veronesi et al., 2005). Increased breast cancer awareness with breast self-examinations and major improvements in routine breast cancer screening have significance effects on early detection of breast cancer. The earlier breast cancer is detected, the better the survival rate. At present, mammography is the best accessible method for earliest diagnosis of breast cancer, most treatable stage - an average of 1.7 years before the woman can feel the lump. Treatment is most effective before the disease spreads. When breast cancer is diagnosed at a local stage, the 5-year survival rate is greater than 90%. Current screening and detection methods are simply not sensitive enough to detect formation of metastasis. Currently, molecular oncology is one of the most promising fields, which provides scope for the early diagnosis and accurate staging of women with breast cancer. The advent of highly sensitive, molecular techniques, such as the polymerase chain reaction (PCR), enables the detection of circulating tumor cells and small metastasis at the molecular level. PCR-based assays are used for the 11
detection of tumor cells in lymph nodes, resection margins, bone marrow and blood (Raj et al., 1998). Progression of human cancers due to the gene amplification/overexpression have been shown to serve as molecular markers that have diagnostic, prognostic and therapeutic relevance. Recently, molecular markers are used to diagnose the occult metastases in cancer patients at early stage and its also better than conventional staging techniques to detect cancer. Several proteins have been found to be specifically overexpressed in certain types of tumors i.e. Her2neu, PSA, p53, pRB, melanoma antigens, etc. (Osman et al., 1999; Russell et al., 2003; Sturgeon et al., 2009). Using sensitive molecular techniques for detection and thus quantification of potential tumor markers could assist in the cancer early diagnosis as well as in the efficacy of anti-cancer therapy. The ideal marker would be useful in diagnosis, staging and prognosis of cancer, provide an estimation of tumor burden, and serve for monitoring effects of therapy, detecting recurrence, localization of tumors and screening in general populations. Some widely used tumor markers are: AFP, Her2/Neu, beta-HCG, CA 19-9, CA 2729 (CA 15-3), CA 125, CEA, and PSA (Zhou et al., 2002; Malati, 2007; Sturgeon et al., 2009). 1.9.2. Biomarkers Biomarkers defined as characteristics that are objectively measured and evaluated as an indicator of normal biological processes, pathological processes, or pharmacological responses to a therapeutic intervention (Srivastava & Srivastava, 2005). The ideal biomarker should have high sensitivity and specificity for diagnosis (Aebersold, 2005) and its level should correlate with disease stage and response to treatment. Molecular oncology is now becoming most promising field that is useful for the diagnosis of breast cancer and its metastases. Tumor marker is a substance present/overexpressed in or produced by a tumor (tumor derived), or the host (tumor-associated), that can be used to differentiate the neoplastic from normal tissue (Zhou et al., 2002; Malati, 2007; Pratheepa et al., 2012). Biomarkers used for cancer screening need to be able to detect early stage of disease with high sensitivity and specificity. However, early detection is only useful if there is an intervention that can be undertaken, when early stage disease has been 12
diagnosed, which results in improved survival outcomes for patients. In addition, widespread screening of the population is not cost-effective when considering cancers that have a low prevalence. Tumor cells grow and multiply, some of their molecules highly produced in tumor tissues and/or released into the blood stream or other body fluids. Therefore, the body fluids or tissues may reflect abnormalities or pathological states that affect the organs and tissues. Tumor markers are found in cells, tissues, and various body fluids such as cerebrospinal fluid, serum, plasma, amniotic fluid and milk (Yu & Diamandis, 1995). The identification or measurement of these ideal markers is useful for diagnosis, staging and prognosis of cancer, which provides estimation for tumor burden, and thus serves for monitoring the effects of therapy, detecting recurrence, localization of tumors and screening in general populations (Pamies & Crawford, 1996). Most of the tumor markers do not fit the ideal profile, because of relative lack of sensitivity and specificity of the available tests. The new biomarkers for cancer patients should be advantageous in population screening for the early detection of cancers, pathological diagnosis, assessment of prognosis, tailoring treatment to individuals, and for assessment of treatment response. With this in mind, circulatory miRNA approaches can be used as biomarkers which could potentially aid prognosis and predict response in breast cancer. One of the major challenges in cancer research is the identification of stable biomarkers, which can be routinely measured in easily accessible samples (Brase et al., 2010). Advanced molecular oncology approaches are urgently needed for the diagnosis of breast cancer so as to evolve treatment strategy to decrease breast cancer caused mortality. Over many decades, many researchers have reported that circulating nucleic acids such as cell-free DNA and RNA are present in serum and other body fluids, which would represent prospective biomarkers (Lagos-Quintana et al., 2001). Present study attempts to improve the diagnosis of breast cancer disease through the development of reagents and protocols for the use of molecular biological advances and the evaluation of relative potential of these diagnostic procedures for the detection and quantification of specific miRNA tumor markers.
1.10. MicroRNA (miRNA) The primary role of RNA was believed to carry genetic information for protein translation. The discovery of small non-coding RNAs and other noncoding RNAs has made many to rethink the role of RNA in biology. In recent years, evidences have accumulated that small non-coding RNAs are also used in a conserved manner to regulate key developmental events (Zhao & Srivastava, 2007). At least four classes of regulatory small non-coding RNAs have been described (Figure 1.5), including microRibonucleic Acid (micro(mi) RNA), short interfering RNAs (siRNA), repeat-associated small interfering RNAs (rasiRNAs) and piwiinteracting RNAs (piRNAs) (Kim, 2006). Among these, small RNAs, miRNAs are the most phylogenetically conserved and which function posttranscriptionally to regulate many physiological processes (Lee et al., 1993; Zhao & Srivastava, 2007). miRNAs belong to a large family of small, single-stranded 16-21 nucleotide long, highly conserved non-protein-coding RNA (ncRNAs) molecules that are master regulators pervasive in higher eukaryotic gene expression at the post translation level of protein-coding genes. Through the binding to complementary sequences in the 3‟ UTR of multiple target messenger RNA (mRNA) and promote their degradation and/or translational inhibition (Pritchard et al., 2012), which are crucial regulators of gene expression in animals, plants and protozoans. miRNAs have different expression patterns to regulate various imperative developmental and physiological processes. There are evidences that miRNAs have been implicated in various biological processes including cell proliferation, apoptosis during development, cell-cell interactions during development of the peripheral nervous system (Stark et al., 2003), to stress resistance and fat metabolism (Xu et al., 2004), from cellularization and segmentation on embryos (Leaman et al., 2005) to cardiogenesis (Kwon et al., 2005) and muscle growth (Sokol et al., 2005), signaling, cell fate identity, organ differentiation and development, stress responses and carcinogenesis (Carrington et al., 2003; Pritchard et al., 2012; Yang et al., 2013).
Figure 1.5. Schematic overview of RNA family 1.11. Discovery of miRNAs From the University of Massachusetts Medical School, developmental biologist, Professor Victor Ambros and his colleagues in 1993 identified miRNA in Caenorhabditis elegans (Nematode) during their genetic study of heterochronic gene 22 nucleotides long lin-14 (Lee et al., 1993). Shortly after its discovery in C. elegans, let-7 had been identified in humans, Drosophila melanogaster, and in several other bilaterian animals, and many other miRNA genes have been discovered in a wide range of species (Lagos-Quintana et al., 2001; Lau et al., 2001; Lee et al., 2001). Currently, computational approaches predicted the existence of approximately 1000 miRNAs in the human genome (Berezikov et al., 2005). In 2006, miRBase was established to provide a database of miRNA sequences and to organize nomenclature (Griffiths-Jones et al., 2006). One thousand and thirty-seven human miRNAs were annotated at miRBase in March 2011. It is believed that miRNAs provide an additional layer for regulation in the cell (Filipowicz et al., 2008). In 2001, interest in the field increased when hundreds of miRNAs were 15
discovered in Drosophila, C. elegans and mammals (Lau et al., 2001). Number of miRNAs has been identified in a wide range of species such as plant (Allen et al., 2004), nematodes, fruit flies, viruses and in humans, further many more miRNA genes are expected to be discovered in many species (Bartel et al., 2004). No miRNAs have been found in yeast and bacteria. It is generally believed that the emergence of miRNAs in evolution is an on-going process (Hertel et al., 2006). The discovery of miRNAs is one of the major developments in molecular biology during the recent decades which has added another dimension to study the regulation of gene expression (Ha, 2011) (Figure 1.6).
Figure 1.6. miRNA research timeline To date, approximately 1,000 different mature miRNAs have been reported in humans. A single miRNA may control hundreds of target mRNAs and hundreds of miRNA genes are predicted and these influences may have consequential effects on gene expression networks (Pritchard et al., 2012). For majority of individual miRNAs the function remains unknown. Particular miRNAs are frequently expressed only in specific cell types or in developmental stages. Numbers of miRNAs have been identified in a wide range of species in plants, nematodes, fruit flies, viruses and human (Tili et al., 2008). Recent studies have also provided evidence that abnormal expression of specific miRNAs is implicated in a number of human diseases, including cancer (Trang et al., 2009).
1.12. Biosynthesis of miRNA miRNA gene transcription takes place within the nucleus, following the cleavage of the ~80 nucleotide stem-loop pre-microRNA precursor performed by the microprocessor complex consisting of Drosha, an RNase III-type nuclease and a double-strand RNA-binding protein co-factor, DiGeorge syndrome critical region 8 gene (DGCR8) in humans. The parturient pri-miRNAs are processed into 60-70 nucleotide hairpin structure (pre-miRNAs) and are exported from the nucleus to the cytoplasm supported by nucleocytoplasmic shuttle protein Exportin-5 in a Ran-GTP dependent manner.
Figure 1.7. Biosynthesis of miRNA (Keerthana et al., 2013) Pre-miRNAs are further cleaved, into an asymmetric duplex by the action of Dicer and accessory proteins Transactivation-responsive RNA-binding protein (TRBP) and PACT in humans, to remove the loop sequence by forming a short-lived asymmetric duplex intermediate (miRNA: miRNA), with a duplex about 22 nucleotides in length. This precursor is cleaved to generate ~21–25-nucleotide mature miRNAs (Figure 1.7). The mature miRNA is loaded into the microRNA-induced silencing complex (miRISC), which binds to target mRNA resulting in either degradation of mRNA, or to blockage of translation without mRNA degradation (He et al., 2004; Lawrie et al., 2010). 17
1.13. miRNA gene regulation in cancer The human genome contains more than 1000 miRNAs, and each miRNA can regulate hundreds of genes, regulating important cellular process such as cell cycle, proliferation, apoptosis, differentiation and development (Johnson et al., 2007; Hermeking, 2009; Wang & Olson, 2009; Broderick & Zamore, 2011). For majority of individual miRNAs the function remains unknown. Particular miRNAs are frequently expressed only in specific cell types or developmental stages. Inappropriate miRNA expression has been linked to a variety of diseases (Broderick & Zamore, 2011). Recent studies have also provided evidence that aberrant expression of specific miRNAs is implicated in a broad spectrum of human diseases, including diabetes, cardiovascular disease and cancer (Esquela-Kerscher & Slack, 2006; Pandey et al., 2009; Zorio et al., 2009). Recent multifarious growing body of evidence suggests that in many human cancers, the miRNAs are aberrantly expressed and several miRNAs have been found to have strong association with oncogenesis and cancer progression. Calin et al. (2002) have first reported the miRNA link with cancer. The potential link between miRNAs and cancer attracted attention soon after the discovery of miRNAs. A large number of miRNAs have been recently implicated in cancer metastasis (Nicoloso et al., 2009). The link between miRNAs and cancer has caught the attention of researchers to elucidate the molecular mechanism of gene regulation (Findlay, 2010). The studies answered a plethora of knowledge that links miRNA expression profiles with specific types of cancer, tumor subtypes, and even metastatic signatures. In addition, functional studies have identified specific miRNAs as tumor suppressors and oncogenes and have linked their expression in breast cancer with the translational regulation of direct mRNA targets. Cancers, being one of the biggest challenges to the world, are also demonstrated to be associated with miRNA dysregulation (Croce, 2009). With identification of more than 1000 miRNAs in humans, it has been estimated that around 30% of the human genes are being regulated by theses miRNAs (Bentwich et al., 2005; Lewis et al., 2005). Some of which are expressed in a tissue-specific and developmental stage-specific manner. For few miRNAs whose function has
been uncovered, they are important regulators for various aspects of developmental control in both plants and animals (Meister & Tuschl, 2004). For majority of individual miRNAs the function remains unknown. Each miRNA has the potential to target hundreds of mRNAs and one mRNA can be targeted by multiple miRNAs (Lim et al., 2005), highlighting the importance of miRNAs in complex networks of gene expression regulation. Moreover, miRNAs shed new light on the posttranscriptional regulation of gene expression. miRNA targets specific mRNAs for destruction (Selbach et al., 2008), causing an active gene to be down-regulated and in some cases to be knocked out completely in its expression. miRNA-mediated repression of target genes differs from conventional regulation mediated by protein factors in many aspects. Each miRNA can target numerous (100-1000) mRNAs, as partial complementary between miRNA sequences and target sites are adequate for interaction (Findlay, 2010). miRNAs frequently act as attenuators rather than on-off switches on gene expression (Vactor & McNeill, 2010; Huang et al., 2013). To date, the functional role of miRNAs in the cell and evolutionary role of miRNAs in species are still poorly understood. Human miRNAs are distributed in all chromosomes except the Y chromosome (Kozomara & Griffiths-Jones, 2010). Initially, miRNAs were thought to be located only within intergenic regions (Lau et al., 2001; Lee et al., 2001). However, Rodriguez et al. (2004) reported that around 70% of mammalian miRNAs are located in defined transcription units (TU) i.e., intronic and exonic miRNAs. Among those miRNAs, 61% RNAs are located in the introns of protein-coding TU, 18% are located in the introns of non-coding TU and some are found in exons of non-coding gene (20%). Given their recognized importance in gene regulation, a link between miRNAs and several major diseases are expected. With the wide impact of miRNAs on gene expression, it is not surprising that a number of miRNAs have been implicated in cancer (He et al., 2005; Lu et al., 2005). The potential link between miRNAs and cancer attracted attention soon after the discovery of miRNAs. Calin et al. (2002) was the first person who explained the link between miRNAs and cancer, when it was discovered that two miRNA genes, miRNA-16-1 and miRNA-15a, are located
in a region on chromosome 13q14,
that is deleted in over 65% of chronic
lymphocytic leukaemia (CLL) patients. Strikingly, half of the known miRNAs are located inside or close to fragile sites and in minimal regions of loss of heterozygosity, minimal regions of amplifications, and common breakpoints associated with cancer. For example, the miRNA cluster 17-92 is located at 13q31, a region commonly amplified in lymphomas (Ota et al., 2004). Enhanced proliferation and dysregulated cell death are the hallmark traits of cancer cell. miRNA can undergo aberrant regulation during carcinogenesis, and they can act as oncogenes (Voorhoeve et al., 2006) or tumor suppressor genes (Michael et al., 2003) (Table 1.1). A growing number of direct and indirect evidences suggest that there exist a relationship between altered miRNA expression and cancer. These include miRNA15a and miRNA-16-1 in CLL (Calin et al., 2004a, b), miRNA-143 and miRNA-145 in colorectal cancer (Michael et al., 2003), let-7 in lung cancer (Johnson et al., 2005) and miRNA-155 in diffuse large B cell lymphoma (Eis et al., 2005). Type of cancer Breast cancer Cervical cancer Pancreatic cancer Brain cancer Lung cancer
Up Regulated miRNA-21
Down Regulated let-7
miRNA-155 miRNA-206 miRNA-210 miRNA-200a
miRNA-10b miRNA-125a miRNA-145 miRNA-203
miRNA-21 miRNA-199a miRNA-196a miRNA-190 miRNA-221 miRNA-21
References Lu et al. (2005) Iorio et al. (2005) Iorio et al. (2005) Volinia et al. (2006) Hu et al. (2010); Pereira et al. (2010) Lui et al. (2007) Pereira et al. (2010) Zhang et al. (2008) Zhang et al. (2008) Zhang et al. (2008) Ridzon et al. (2005) Ridzon et al. (2005) Yanaihara et al. (2006) Megiorni et al.(2011); Landi et al. (2010)
Table 1.1. List of some deregulated miRNAs in different cancers
Expression profiling has identified other cancers with differential expression of several miRNAs, including breast cancer (Iorio et al., 2005), glioblastoma (Chan et al., 2005) and papillary thyroid cancer (He et al., 2005). A polycistron encoding five miRNAs is amplified in human B-cell lymphomas and forced expression of the polycistron along with c-myc which was tumorigenic, suggesting that this group of miRNAs may function as oncogenes (He et al., 2005). Thus, the potential of miRNA research is versatile. With this advanced tool, scientists and clinicians can able to focus not only just finding better treatments but also on finding cures of many diseases ailing in the world today, including viral, cancer, cardiovascular, neurodegenerative, inflammatory and metabolic diseases. Modern biomedical science is finally bringing together the intellectual forces of international academic researchers, industry scientists, and clinicians and thus collaborations are of high relevance for emerging science of miRNAs research, which holds such a great therapeutics potential and understanding of development of diseases and their treatment method. 1.14. Dysregulation of miRNA in breast Cancer miRNAs are aberrantly expressed in several cancers, including breast cancer and that some miRNAs have the potential to act as tumor suppressors or oncogenes (Wiemer, 2007; Schaefer et al., 2009). Several miRNAs have been found to be deregulated in breast cancer (Blenkiron et al., 2007) (Figure 1.8).
Increased expression of OncomiR
Decreased expression of tumor suppressor miRNAs
miRNA-21 miRNA-155 miRNA-206 miRNA-210 miRNA-10b miRNA-125a miRNA-126 miRNA-145 miRNA-205 mi NA-210
Figure 1.8. Breast cancer associated oncomiRs and tumor suppressor miRNAs
A large number of miRNAs have been recently implicated in cancer metastasis (Nicoloso et al., 2009), miRNAs are also aberrantly expressed in human breast cancer. Example, some miRNAs are downregulate expression such as: miRNA-10b, miRNA-125b, miRNA-145, miRNA-126, miRNA-335, miRNA-373, miRNA-146, miRNA-520c and miRNA-205 emerged as the most consistently deregulated in breast cancer. The miRNA-21 and miRNA-155, were upregulated (Iorio et al., 2005; Lee et al., 2007; Wu et al., 2009) (Table 1.1). 1.15. Role of miRNA in medicine 1.15.1. Improvement of existing miRNA therapies The discovery of the miRNAs role in carcinogenesis has propelled miRNAs to become focal targets for early diagnosis and then for novel therapeutic approaches and thus efforts have been made in recent years to develop miRNAbased therapeutics. Delivery of RNA-mimies or antagomiRs is a critical issue for effective therapy (Selvamani et al., 2012). In recent years there has also been an explosion of research reports on miRNA myriad role in biomedical fields, as master regulators of the human genome (Bartel et al., 2004). miRNA deficiencies or, abundances due to the single point mutation or epigenetic silencing, of the abnormal expression level have been correlated with a number of clinically important patho-physiology of diseases and their status, to become important diagnostic and prognostic tools. (Soifer et al., 2007). They play crucial roles in a wide range of tools of medicine for prevention, diagnosis, prognosis and therapy of human diseases (Hesse et al., 2013 ). miRNA expression can be appropriately linked to a variety of diseases including cancer. In cancer, miRNA can control multiple oncogenes (Dvinge et al., 2013) and oncogenic pathways (Bader et al., 2011). Currently used body fluid-based diagnostic methods exhibit low sensitivity and specificity which limits their clinical application (Rupani et al., 2012). After the discovery of circulating miRNAs, the potential application as powerful biomarkers for disease diagnostics, monitoring therapeutic effect and predicting recurrence in many diseases including cancers are promising. Circulatory miRNA biomarkers are more attractive diagnostic tools because they are remarkably stable in body fluid against endogenous RNase activity, easily 22
accessible to the body fluids and easily detectable in plasma, serum, saliva, sputum (Leidner et al., 2013; Heneghan et al., 2010) and urine samples (Mitchell et al., 2008). It was confirmed by Ho et al. (2010) who have reported that statistically significant elevated plasma levels of miRNA-210 in pancreatic cancer patients compared with age matched healthy controls, using qRT-PCR. It may potentially serve as a useful biomarker for pancreatic cancer diagnosis. Huang et al. (2010) found that plasma miRNA-29a and miRNA-92a are potential novel non-invasive biomarkers for early detection of CRC. To further explore the origins of these circulating miRNAs, Heneghan et al. (2010) have studied a panel of 7 candidate miRNAs which were quantified in tissue and blood specimens of 148 breast cancer patients and 44 age matched disease free controls. They found that miRNA-195 was significantly over expressed in the circulation of breast cancer patients, moreover the miRNA-195 expression level decreased in the post-operative period of the same patients. Cardiac specific miRNA-208a expression levels were elevated during analysis of plasma samples of acute myocardial infarction (AMI) but additionally the miRNA was absent in the plasma samples of healthy people. Thus, the miRNA208a is considered as a novel biomarker for early detection of myocardial injury in humans (Wang et al., 2010). Furthermore, serum miRNAs could also be useful biomarkers for diagnosing several human diseases. In a previous study in serum samples of sepsis patients, miRNA-146a and miRNA-223 were used as serum biomarkers for the diagnosis of sepsis (Wang et al., 2012). Zhao et al. (2012) reported pregnancy-associated circulating miRNA-323-3p which significantly increased in maternal circulation during pregnancy and hence proposed as a potential biomarker for the diagnosis of pregnancy-associated complications. miRNA-451 has 50 fold over expression in maternal plasma of pregnant women with twins in relation to single pregnancy (Ge et al., 2011). The recent observations on endothelial miRNA-126 are deregulated in patients with type 2 diabetes (DM), which could be used as a biomarker for early detection of vascular complications of diabetes (Zampetaki et al., 2010) and as a realizable RNA-based therapeutic agent for diabetes-induced atherosclerosis (Fernandez-Valverde et al., 2011). After exposure of ionizing radiation both in vitro and in vivo models, the miRNA-34a expression level has been found to be elevated 23
(Tili et al., 2008). Recent studies support that, the exciting prospects of utilizing circulating miRNAs as non-invasive, sensitive biomarkers for cancer (breast, cervical, pancreatic, liver, lung, colon and prostate cancer) and other diseases (Leidner et al., 2013). 1.15.2. miRNAs as blood based biomarkers The most used current diagnostic cancer biomarkers are effortful and laborintensive procedures and several markers can be tested for at a time. Therefore, to develop conventional blood based biomarker, due to the
availability of blood
sample, simply diagnostic biomarkers found in blood stream would be alternative to traditional methods of diagnosing for cancer (Lena et al., 2008). Minimal invasive biomarkers development for early detection of the breast cancer is really important for patients outcomes and survival, reducing patient suffering and to significantly reduce the financial costs for the diagnosis and treatment. Therefore, more accurate and powerful diagnostic and predictive tools are needed for non-invasive breast cancer. So, now the biomarker research is one of the most rapidly growing areas in the field of circulating nucleic acids in plasma and serum. Determining the expression ratios of genes or miRNAs has been considered to be a useful technique to improve the diagnostic potential (Tsujiura et al., 2010). Recently it has been proved that the circulating miRNAs are relatively stable, very accessible, low invasive, easily testable, promising and stage-specific biomarkers for non-invasive diagnosis in various tumors (Brase et al., 2011; Etheridge et al., 2011). Researchers have shown that miRNAs as a remarkably stable in blood stream against endogenous RNase activity, which can act as a novel promising blood-based biomarker for cancer detection and diagnosis (Mitchell et al., 2008, Fu et al., 2011; Wu, 2012). It has been observed that miRNA expression is generally higher in tumor compared to normal tissues that led us to the hypothesis that global miRNA expression reflects the state of cellular differentiation. miRNAs are released from tumor tissues to bloodstream or other body fluids. Cell-free miRNAs are highly stable in human blood stream (identified in plasma and also serum) against endogenous RNase activity. The level and specificity of miRNAs in body fluids may reflect abnormalities or pathological 24
condition affecting organs, tissues/cancer and other disease states. Therefore, based on elevated level miRNA are measured from this body fluids, which develop a novel specific and very sensitive blood-based diagnostic tool for early detection of cancer (Alton et al., 2011; Maria et al., 2011). Differentiatl miRNA expression profilies of normal and cancer tissue can be assessed through a unique miRNA expression signature by microarrays and other convenient methods. These profiles can be related to different tumor types and tumor grades. The convinced miRNA signatures are used for prognosis that could be used to determine the specific course of treatment (Trang et al., 2011). Cancer-specific miRNAs are shedding light onto the molecular basis of cancer, being able to identify cancer-specific expression of miRNAs in blood which is playing vital role in early-stages diagnosis of cancer. Currently blood based biomarkers are widely used for diagnosis of diseases and in response to treatment (Allen et al., 2004). The expression profile number of miRNAs are differentially up regulated or down regulated in the tumors and cell lines compared to the normal breast tissue (Iorio et al., 2005; Lu et al., 2005). One miRNA targets multiple mRNAs, one deregulated miRNA in cancer can affect the expression of scores of downstream genes. The expression of miRNA is deregulated in many types of cancer, indicating that miRNAs play important role in cancer (Giglio et al., 2010). The expression profiles of miRNAs are reported to be tissue-/cell-specific (LagosQuintana et al., 2002 & 2003). Expression profile of miRNA can directly reflect the disease status (Lu et al., 2005). miRNA profiling of human cancers is a useful tool to classify cancer type according to their origin, because of its tissue-specific nature. The miRNA profiling can distinguish the phenotypic signature of breast cancers and that miRNA can be used as a potential diagnostic marker. Screening study to identify the circulating miRNAs (miRNAs-21 & 146a) in relation to breast tumor progression is an important clinical relevance.
1.15.3. Therapeautic for cancer management Recently, most of the reported miRNAs are being used for potential therapeutic approach like antisense-mediated inhibition of oncogenic miRNAs. In cancer cells some specific miRNAs are frequently overexpressed and most miRNAs are downregulateds (Kota et al., 2009). Globally, miRNA repression enhances cellular transformation and tumorigenesis in both in vitro and in vivo models (Kumar et al., 2007), underscoring the protumorigenic effects of miRNA loss-offunction. In miRNA-based therapeutics, there are two approaches: miRNA suppression and miRNA mimics. miRNA suppressions are generated to use small molecule inhibitors or siRNAs that inhibit specific endogenous miRNAs which show a gainof-function in diseased tissues. miRNA mimics (reexpression) are used to restore the lost function. This approach, also known as „miRNA replacement therapy‟, reintroduce miRNAs into diseased cells that are normally expressed in healthy cells (Bader et al., 2010). Kota et al. (2009) studied that systemic therapeutic delivery of miRNA-26a in a mouse model of hepatocellular carcinoma (HCC) using adeno-associated virus (AAV). This miRNA-26a is expressed in high levels in normal adult liver tissues, in case of
HCC cells exhibit reduced expression levels. The miRNA-26a directly
downregulates cyclins D2 and E2 and induces a G1 arrest in human liver cancer cells in vitro. Observation of this results, AAV-mediated miRNA-26a delivery potently inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and dramatic protection from disease progression without toxicity. Trang et al. (2011) have shown that therapeutic exogenous delivery of let-7 to established tumors in KRAS-G12D transgenic mouse models of non-small-cell lung cancer (NSCLC) significantly reduces the tumor burden. Similiary, for NSCLC, therapeutic formulation using chemically synthesized miRNA-34a and a lipid-based delivery vehicle had blocked the tumor growth in mouse models. The therapeutic efficiency correlates with reduced prliferation and enchanced apoptotic activity of tumor cells, as well as a specific repression of CDK4, MET and BCL-2 (Wiggins et al., 2010). Trang et al. (2011) have made similar interesting study during wich they
have systematically delivered the synthetic tumor supressor miRNA mimics in complex with a novel neutral lipid emulsion to autochthonous mouse model. In lung cancer both miRNA-34a and let-7 are frequently down regulated. Delivered mice with miRNA-34a exhibited a 60% reduction in tumor area compared to mice treated with a miRNA control (Trang et al., 2011). The findings of more recent research provide scope for the development of sophisticated miRNA-based cancer therapy. Yu et al. (2010) have reported ectopic expression of miRNA-96 through a synthetic miRNA precursor inhibited the KRAS oncogene and the result in decreased cancer cell invasion, migration and thus reduced the tumor growth in pancreatic cancer cells, and it provides a novel therapeutic strategy for treatment of pancreatic cancer. There exists another interesting study by Kota et al. (2009) in the murine liver cancer model of HCC. Their systemic administration of miRNA-26a using an AAV effects in inhibiting cancer cell proliferation, induction of tumor-specific apoptosis, and effective protection from disease progression without toxicity. Above observed evidences suggests that miRNAs are being found as promising agents in cancer therapy. Recent therapeutic strategies not only have focused on the regulation of miRNA function but also have tried to improve the existing therapies. For example, glioma cells can be sensitized by treatment with 5-fluoroacyl if it is simultaneously delivered to cells with miRNA-21 (Ren et al., 2010). Cells can also be sensitized to doxorubicin if they are cotransfected with TP53 and a synthetic p21-targeting miRNA (Idogawa et al., 2009). Moreover, miRNA-521 can sensitize cancer cells to radiation (Josson et al., 2008). The specificity of viral-based treatments can be enhanced by introducing miRNA binding sites to the viral genome, thereby viral replication is prevented in all cells but the target cells. The introduction of miRNA145 and miRNA-143 binding sites downstream of the herpes simplex virus ICP4 gene restricts oncolytic activity to prostate cancer cells where expression of this miRNA is lost (Lee et al., 2009a). A similar approach has been administered to improve the specificity of an oncolytic virus in liver cancer (Cawood et al., 2009). Four binding sites of the liver-specific miRNA-122 were introduced downstream of the EA1 binding cassette in a cytotoxic liver adenovirus.
Based on the above literatures and findings the miRNA studies strongly supports the systemic delivery of tumor-suppressing miRNAs as a powerful target for anticancer therapeutic modality. 1.16. Review of literature 1.16.1. Circulatory miRNA as a biomarker Recent studies have shown that specific cancer characteristics, both genetic and epigenetic, are detectable in the plasma and serum of cancer patients and which could be useful as a tool for early diagnosis of cancer disease. It has been reported that the circulating nucleic acids such as cell-free DNA and RNAs are present in serum and other physiological body fluids, which may represent prospective biomarkers (Tsang & Lo, 2007). Analyses of molecular genetic markers in biological fluids have been proposed as a useful tool for cancer diagnosis. Existing biomarkers for breast cancer have many inherent deficiencies. Presently, breast cancer screening technique like histological evaluation of tumor material obtained from tissue biopsy, remains the gold standard for diagnosis (Wittmann & Jack, 2010). While mammography is being currently considered to be the gold standard diagnostic tool, it is not without limitations, including use of ionizing radiation and a considerable false positive rate of 8-10% (Taplin et al., 2008). Although tumor markers could greatly improve the diagnosis, the invasive, unpleasant, and inconvenient nature of current diagnostic procedures limits their clinical application. Thompson et al. (2008) have reported, that to date, only two biomarkers have been established in the routine assessment of breast cancer: ER and
epidermal growth receptor 2 (HER2). Although these markers are currently available, ER and HER2 assessment is far from perfect (Gebhart et al., 2005). A number of circulating tumour markers (e.g., carcinoembryonic antigen (CEA) and carbohydrate antigen 15-3 (CA 15-3) are used clinically for the management of breast cancer, but, CA-125 remains a poor marker for early stage disease and has a documented sensitivity of 40% and hence, they are not used as screening tools (Harris et al., 2007) though they have been in clinical use as prognostic markers. Despite their frequent use, CEA and Ca 15.3 remain poor markers for early stage 28
disease with a documented preoperative sensitivity of only 9.11 and 5.36 respectively (Uehara et al., 2008). Therefore, it would be highly desirable to develop non-invasive tumor markers. Tumor-associated RNAs have been described in the serum/plasma of cancer patients for more than a decade (Tsang et al., 2007). More recently, several reports also showed that circulating miRNAs existed in serum/plasma (Mitchell et al. 2008). miRNAs are a class of small RNA molecules with regulatory function, and play an important role in tumor development and progression (Hammond et al., 2006). It has been demonstrated that tumor-derived miRNAs exist in the circulating nucleic acids of cancer patients. Tumor miRNAs may be present as a result of tumor cells dying and getting lysed or tumor cells releasing miRNAs into the surrounding environment. This phenomenon implies that detection of the circulating miRNA may be an effective method for non-invasive diagnosis of cancer biomarker (Calin et al., 2006; Garofalo et al., 2008). Tumor-derived miRNAs was first described in plasma by Mitchell et al. (2008). They found that plasma miRNA-141 could efficiently identify prostate cancer patients, and this circulating miRNAs may have important value for cancer diagnosis. Circulatory cell free miRNAs have been demonstrated to be a more accurate method for classifying cancer subtypes than circulatory cell free DNA and RNA. Due to high stability of miRNAs in clinical source such as sputum, plasma, serum, urine, saliva etc., (Wittmann & Jack, 2010), miRNAs have great potential to be used as diagnostic and prognostic markers for cancer, diabetes mellitus, pregnancy and other diseases (Mitchell et al., 2008). But due to their small size, accurate and sensitive detection of miRNAs is highly challenging. The first evidence that miRNAs exist and can be detected outside living cells has been provided by Valadi et al. (2007). Chim et al. (2008) screened plasma samples for the presence of four particular miRNAs enriched in the placenta, and found that three of them present in high concentrations specifically in maternal, but not from postpartum, plasma. One of them, miRNA-141, seemed to increase with gestational age, reaching its greatest abundance in the third trimester, but it was absent in control male sera. Aslam (2010), identified cancer related miRNAs in the
circulating blood at levels sufficient to be measured for the detection of tumours. This feasibility study demonstrated the potential use of circulating miRNAs for the early detection of breast cancer (Mitchell et al., 2008). Recently, a number of researchers have examined plasma miRNA concentrations from patients with gastric cancer-miRNA-17-5p, miRNA-21, miRNA-106a, miRNA-106b (Schetter et al., 2009; Tsujiura et al., 2010), colorectal cancer- miRNA-92 (Huang et al., 2008; Ng et al., 2009), oral squamous cell carcinoma-miRNA-31 (Liu et al., 2010), pancreatic cancer- miRNA-210 (Ho et al., 2010), prostate cancer-miRNA-141 (Mitchell et al., 2008) miRNA-195 and let-7a in breast cancer (Heneghan et al., 2010) and miRNAs were found to be significantly higher than controls, and hence, there is a need to assess their clinical application for diagnosing and monitoring of diseases. Croce et al. (2005) first observed the significant differences in the expression of miRNA profile between human B cell CLL and normal CD5-B cells using a microarray, Northern blot analyses and qRT-PCR. Ho et al. (2001) extracted miRNA-210 directly from pancreatic cancer and age-matched non cancer controls plasma, and he opined that miRNA-210 may serve as a diagnostic marker for screening or surveillance for pancreatic cancer by using reverse-transcribed cDNA. Cheng et al. (2013) have proved that cell-free miRNAs are highly stable in human blood stream (identified in plasma and serum) against endogenous RNase activity; they also distinguished differential expression of miRNA-141 level in prostate cancer plasma in relation to normal. 1.16.2. miRNA-21 miRNA-21 is also known as hsa-miR-21; and the gene is located in an intergenic region. The length of miRNA-21 gene is reported as 3433 nucleotides long. The mature miRNA forms one strand of the RNA duplex. One strand is degraded and other is incorporated in to a protein complex, RNA induced silencing complex (RISC), targets a partially binding complementary target mRNA. The human miRNA-21 gene is located on plus strand of chromosome 17q23.2 (55273409 - 55273480) within a coding gene TMEM49 (also known as
Human Vacuole Membrane Protein1-VMP-1). miRNA-21 has been linked to a variety of diseases, including cancer, fibrosis and heart disease and is therefore a potential target for a number of therapeutic indications. Generally, miRNA-21 is overexpressed in most human cancers and which down regulates the expression of many tumor suppressors. There are many cancer control genes that are targeted by miRNA-21 (Wickramasinghe et al., 2009). miRNA-21 interestingly was found to be the one that is a commonly over expressed miRNA. miRNA-21 has attracted more attention than any other miRNA, because it is one of the most highly up-regulated genes in various solid tumor tissues (e.g., glioma, colorectal cancer, stomach/gastric cancer, hepatocellular carcinoma, pancreas cancer, lung cancer, cholangiocarcinoma, leukemic cancer, ovarian cancer, esophageal cancer (Feber et al., 2008), uterine leiomyomas (Wang et al., 2007), head and neck cancer (Tran et al., 2007), colorectal cancer (Slaby et al., 2007) prostate cancer (Krichevsky & Gabriely et al., 2009) and highly upregulated in breast cancer (Meng et al., 2007), cardiac hypertrophy and neointimal formation, suggesting that it has a fundamental role in cell growth. miRNA-21 has been found to play an important role in cancer development and progression via: regulation of transformation, invasion, metastasis, cell growth and apoptosis. Thus for a start it seems promising to study the effect of inhibition as well as overexpression of miRNA-21 in Chinese Hamster Ovary (CHO) cells under various conditions. The first serum miRNA biomarker discovered was miRNA-21, and patients with diffuse large B-cell lymphoma had high serum levels of miRNA-21 (Lawrie et al., 2008). Li et al. (2009) have first reported the miRNA profiling in human prostate cancer cell lines: DU- 145, PC-3 and LNCaP and they found that miRNA21 was over expressed. Therefore, they opined that miRNA-21 plays an important role in apoptosis and metastasis of prostate cancer. The impact that increased levels of miRNA-21 in cancer tissues have seem to be manifold: in glioblastoma cells miRNA-21 was reported to be highly expressed; upon specific miRNA-21 knockdown increased occurrence of apoptosis was observed due to higher caspase activity (Chan et al., 2005) and which sensitizes cholangiocytes to chemotherapeutic agents (Meng et al., 2007). Whereas, its overexpression inhibits 31
apoptosis in myeloma cells (Loffler et al., 2007). Hence, miRNA-21 role was attributed to the anti-apoptotic activity. It was shown that miRNA-21 is upregulated in glioblastoma and knockdown of miRNA-21 enhanced apoptosis, suggesting that miRNA-21 targets apoptosis-related genes. miRNA-21 has been shown to target and down-regulate the expression of the tumor suppressors tropomyosin 1(TPM1) (Si et al., 2007), PTEN (Meng et al., 2007), programmed cell death 4 (PDCD4), promote cell invasion and metastasis (Asangani & Rasheed, 2008). Moreover, anti-miRNA-21 inhibits tumor growth in vivo and in vitro (Si et al., 2007). In human colorectal cancer, the levels of miRNA21 positively correlated with the development of metastasis but not tumor size (Slaby et al., 2007). Interaction of miRNA-21 with tumor suppressor gene, PDCD4 has been identified in colorectal cancer cell lines, where this interaction was suggested to stimulate invasion, intravasation and metastasis (Asangani et al., 2008). In MCF7(Michigan Cancer Foundation-7) cells, which are a commonly used early breast cancer cell line with high levels of PDCD4 and relatively low levels of miRNA-21, neoplastic transformation has been shown to be influenced by mature miRNA-21 levels (Frankel et al., 2008). Currently, PDCD4 is the most frequently cited target of miRNA-21, and that is why it makes sense to take a closer look on this protein: about a decade ago the mouse mRNA „MA-3‟ was identified to be upregulated during apoptosis in several cell lines such as thymocytes, T cells and B cells (Shibahara et al., 1995). Besides regulation of transformation, invasion and metastasis, the miRNA-21 has also been related to tumor growth in breast cancer tissue. Si et al. (2007) showed that inhibition of intracellular miRNA-21 levels via anti-miRNA-21 transfection, resulted in reduced cell growth in vitro as well as reduced tumorgrowth in a xenograft mouse model. Thus, miRNA-21 has been described as an interesting drug target for cancer therapies. On the other hand it has been found that some cell models seemed to behave quite differently upon miRNA-21 inhibition or overexpression. Similarly, Cheng et al. (2008), reported the effect of several miRNA inhibitors on cell growth and apoptosis of HeLa cells. Here, anti-miR-21
transfection resulted in increased growth in relation to control, but no effect of miRNA-21 knockdown on apoptosis (by measuring caspase-3 activity) was observed (Cheng & Byrom, 2005). The Patel laboratory demonstrated that miRNA-21 is highly expressed in hepatocellular cancer (Meng et al., 2007). Inhibition of miRNA-21 sensitized the effect of gemcitabine and miRNA-21 functioned in cell proliferation, migration and invasion targeting the PTEN of PI3 kinase signaling. Loffler et al. (2007) explained that miRNA-21 contributes to signal transducer and activator of transcription 3 (Stat3) - mediated survival of myeloma cells. Iorio et al. (2005) and Si et al. (2007) have also found that miRNA-21 is overexpressed in breast cancer. Si et al. (2007) explained that anti-miR-21 oligonucleotide decreased cell growth in vitro and in vivo and hence suggested that miRNA-21 is an oncogene that is associated with the anti-apoptotic gene BCL-2. Zhu et al. (2007) used two-dimensional gel electrophoresis to identify targets of miRNA-21 and found that the tumor suppressor gene TPM1 as a target of miRNA-21 in a breast cancer cell line. More recently, miRNA-21 was demonstrated to be widely overexpressed in an array of tumors including those derived from breast, colon, lung, pancreas, stomach and prostate (Volinia et al., 2006; Krichevsky et al., 2009; Meng et al., 2007). The foregoing account provide strong evidence that overexpression of miRNA-21 promotes tumorigenesis by suppressing apoptosis. 1.16.3. miRNA-146a miRNA-146a is located on chromosomes 5q33. Recently, miRNA-146a was shown to inhibit both migration and invasion, suggesting its role in metastasis (Hurst et al., 2009). Recently, a number of researchers have predicted and identified miRNA-146a and its target like IL-1 receptor-associated kinase 1 (IRAK1), IRAK2, TNF receptor-associated factor 6 (TRAF6), RIG-I, IRF-5, STAT-1, PTC1, Numb, interleukin (IL)-8, IL-6, CXCR4, matrix metalloproteinase-9, EGFR, breast cancer metastasis suppressor 1(BRMS1) (Douglas et al., 2009), BRCA-1 and BRCA-2 (Hilton et al., 2002). He et al. (2005) have initially focussed on miRNA-146a in relation to tumourigenesis. They observed that miRNA-146a overexpression has also been 33
linked to papillary thyroid. Bhaumik et al. (2008) have reported that miRNA-146a involved in the ability of BRMS1 to suppress metastasis in breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Therefore, they opined that, miRNA-146a has a promising therapeutic potential to suppress breast cancer metastasis. Richard et al. (2008) found that the genetic polymorphism in the miRNA-146a gene (rs2910164) was associated with young age of familial breast/ovarian cancer diagnosis. miRNA146a can suppress the metastatic ability of breast cancer cells partially through decreasing constitutive NF-κ B activity (Bhaumik et al., 2008). Volinia et al. (2006) analysed the expression of miRNA-146a in breast tissues through microarray-based method and found that the miRNA-146a was significantly upregulated in breast carcinoma tissues in relation to normal tissue. From the foregoing account, it is clear that studies on the use of plasma miRNAs in relation to breast cancer progression, are needed. 1.16.4. Oncogene-KRAS KRAS is frequently affected by oncogenic mutations in colorectal carcinomas and is observed in other tumors such as lung cancer (Benvenuti et al., 2007; Marchetti et al., 2009). Importantly, several studies have indicated that the effectiveness of anti-EGFR drugs is limited to those tumors harboring no oncogenic mutations in KRAS and the presence of mutant KRAS in lung and CRC tumors correlates with poor prognosis (Benvenuti et al., 2007; Fiore et al., 2007). KRAS mutations may mediate resistance to apoptosis induced by chemotherapeutic agents (Patrizio, 2005). Theoretically, KRAS codon-12 mutations could be found more often in tumour tissue because the bases of KRAS codon-12 might be more amenable to damaging events (Kraus et al., 2006). Bazan et al. (2002) described that an association between KRAS codon-13 mutations and the presence of lymph node metastasis. Oncogenic RAS proteins can signal cell proliferation without EGFR activation and thus, molecular testing of human KRAS mutations is of great relevance in the identification of patients and also used in the anti-EGFR therapies. In a study reported by Hollestelle et al. (2007) KRAS mutations were found in 5 out of 40 different breast cancer cell lines (13% incidence). Overall, KRAS mutations
are infrequent in breast cancer, representing a mere 5% of all breast carcinomas (Karnoub et al., 2008). Hollestelle et al. (2007) found mutations in 12.5% of cases but the Sanger COSMIC database version5 records only a 5% incidence (Bamford et al., 2004). The lower frequency of KRAS mutations in breast cancer cell lines suggests that the gene mutation may be less important in carcinogenesis of breast cancer than in other forms of cancer, although mutations at a “hotspot” in the KRAS gene have been found in a small subset of breast cancers. From the above account, it is clear that studies on the KRAS mutation in relation to TNBC progression are needed. The present study deals with the identification of circulator
(miRNAs-21 & 146a) in relation to breast tumor progression. 1.16.5. Anticancer drugs The drugs used to combat cancer belong to one of two broad categories. The first is cytotoxic (cell killing) drugs and the second is cytostatic (cell stabilizing drugs).Cytotoxic drugs effects by interfering with DNA replication. Cytostatic drugs are specifically target the altered biochemical pathways and to deactivate the altered enzymes that result from changes in the oncogene involved (Parihar et al., 2012) (Figure 1.9)
Figure 1.9. Anticancer drugs, their mechanisms and chemical structures
Mhaidat et al., (2007) have reported that docetaxel induces caspase-2dependent apoptosis of melanoma through the mitochondrial pathway. Breast cancer cells (BCap3) treated with
at the ≥5 nmol/L dose has resulted in the
initiation of mitotic arrest by showing DNA laddering and sub-G0 DNA content, which are associated with apoptosis (Zeng et al., 2000). Wang and Wieder (2004) have reported that docetaxel-induced cell death is caused by c-Jun NH2- terminal kinase-mediated apoptosis in breast cancer cells: MCF-7, SK-Br-3, and MDA-MB231. Studies have to be focused on the functions of miRNAs besides improving the existing chemotherapies.The specificity of viral-based treatments can be enhanced by introducing miRNA sites to the viral genome and thereby viral replication can be prevented in targeted cells and such an approach has been administered to improve the specificity of cancer cells to prevent the uncontrolled cell proliferation. Hence miRNA studies strongly advocate the systemic delivery of tumor-suppressing miRNAs as a powerful target for anticancer therapeutic model.
1.17. Scope of the present study The present investigation is mainly aimed to investigate the miRNA expression profile and KRAS mutation analysis in breast cancer samples and cancer cell lines, with the following objectives: • To study the expression profile of circulating miRNAs-21&146a of breast cancer plasma samples vis-a-vis healthy individuals. • To analyse the mutation of oncogene-KRAS in different breast cancer cell lines. • To study the expression pattern of a set of miRNAs (let-7a & g, miRNAs-10b, 21 & 155) and to determine whether there is any potential miRNA that is specifically linked to younger age-breast carcinomas. • To assess the effect of anticancer drugs on the miRNA-21 expression level in breast cancer cell line.