Signalling Pathways

The research paper by Walker, Xiang, and Frank (2014) was evaluated and analysed for the purpose of developing a diagnostic and prognostic tool for breast cancer. The two primary transcription factors used as diagnostic tool against breast cancer are the transducer and activator of transcription (STAT5 and STAT3). These two prime transcription factors were primarily used due to breast cancer cells containing an improper activation of transcription factor involved in the development of mammary cell. Consequently the research article is demonstrating that a prime transcription factor that possesses a significant effect in increasing the development of mammary cell than a diagnostic could be formed for breast cancer.

Signal transducers and activators of transcription (STATs) are latent transcription factors that become activated after tyrosine phosphorylation by receptor and non-receptor tyrosine kinases. After phosphorylation, the STATs become activated, shift to the nucleus, and bind to similar STAT DNA binding places, in which they modify transcription of major target genes convoluted in most of the cellular processes.  STATs are often irrationally active in cancer: STAT5 and STAT3 are usually activated constitutively in breast cancer, in contrast to the transient activation which takes place physiologically (Banerjee and Resat, 2016; Walker et al., 2009). STAT3 and STAT5 are two closely members of the STATs family that have separate functions in the development of the mammary glands, and have also been found to be activated in breast cancer (Brantley and Benveniste, 2008). The key steps of the signalling pathways of the JAK-STAT is shown below. The signalling pathway comprises of three primary proteins: STATS, JAKs, and cell-surface receptors.

             

                                                               Source: (Liongue et al., 2012)

Several studies have been conducted on the relationship between transcriptional factors and breast cancer.  The first initial research developed for signal transcription factors in mammary cell was published by Clarkson et al. (2006) and countless other scientists continued the research to enhance the study and to establish a better therapy for cancer (Yu, Pardoll, and Jove, 2009; Walker et al., 2009; Creamer et al., 2010).  There are minor contrasts between the first initial research (Clarkson et al. (2006) and the research by Walker et al. (2009). The existing difference is that the initial research only focus on the development of mammary epithelial cells using STAT3, STAT5 and Prolactin and Leukemia inhibitory factor (LIF), whereas, the second research focuses on diagnostic and prognostic in mammary gland using STAT3 and STAT5. The research of Sarah R. Walker et al. (2009) is building on the previous work carried out by Richard W.E Clarkson et al. (2006) but aiming at finding a diagnostic in breast cancer, using a different approach in methodology in order to improve the progress. Clarkson et al. (2006) found out that specific activation of STAT3 and STAT5 alone is enough to prompt and subdue apoptosis respectively through the inducement of specific genes in mammary epithelial cells. On the other hand, Walker et al. (2009) found out that STAT5 can modify the impacts of STAT3 beginning from the state of gene expression to cellular phenotype. Walker et al. (2013) conducted a study to ascertain the method by which BCL6 transcription is regulated by the oncogenic transcription factors. The authors found out that STAT3 raises BCL6 expression and improves the recruitment of RNA polymerase II, whereas STAT5 represses expression of BCL6 below basal levels and lowers the association of RNA polymerase II.

Background

Several methods were used by Walker et al. (2014) to provide evidence to their study and hypothesis. These methods included immunohistochemistry, gene expression array analysis, cell lines and stimulations, immunoblots, reports gene assays, reverse transcription PCR, short interfering RNA, viral production and infections, viability assays, and lastly caspace activation assay. These tests were carried out to ascertain the characteristics of tumours showing activation in protein, and also to test the specificity of STAT5 and STAT3.

The effects of the STATs were analysed on gene regulation and phenotype of mammary carcinoma cells (Frasor et al., 2003). The co-activation between the two STATs have played a significant effect on the sensitivity and proliferation, based on the findings of the authors, which show that STATs reduced proliferation and improved sensitivity to the chemotherapeutic drug namely paclitaxel and vinorelbine, in contrast to STAT3 alone or STAT5 alone. Overall STAT5 possesses a dominant role because it can change the effect of STAT3 at different stages of gene expression to cellular phenotype and trigger the activation state of both STATs, which concludes that both STATs are vital in providing a diagnostic and prognostic to breast cancer (Walker et al., 2009).

Signal tranducer and activator of transcription 3 and 5 are transcription factor which in human it’s encoded by the STAT3 and STAT5 gene, hence they are the members of the STAT protein family. STAT3 and STAT5 protein regulate gene associated in survival and proliferation. Therefore, they are very vital as they play a significant role in regulation mammary gland (Clarkson et al., 2006). The two transcription factors have similar formation but they are divergent in terms of phenotype and gene expression. For instance, when STAT5 and STAT3 are activated at the same time, the effect of STAT5 is dominant causing low proliferation and high sensitivity to cell death (Walker et al., 2014).  

Mammary growth takes place through specific activation of many transcription factors. Incorrect activation of most of the transcription factors is common in breast cancer and may directly lead to its pathogenesis (Visvader and Lindeman, 2003). More precisely, STATs have significant functions in most of cellular functions, and in cancer they are often activated incorrectly (Thomas et al., 2015). Studies have shown that STAT5 is inappropriately activated in a subset of breast cancers, commonly in tumours that are well differentiated (Cotarla et al., 2004; Nevalainen et al., 2004). The function of STAT5 in mammary tumorigenesis has also been evidenced using Murine models (Lavnilovitch, Groner, and Barash, 2002); Ren et al., 2002). An in appropriate activated state of STAT5 expressed in mice leads to mammary carcinomas, while the absence of STAT5a in mice leads to the protection of the same mice from mammary tumours caused by transforming growth factor alpha. Therefore, the findings of these authors demonstrate that STAT5 has an important function in both normal and mammary function.

STAT3 has also a central function in the growth of mammary cells, however, with a single discrete role from STAT5. Studies have shown that STAT3 is activated by leukemia inhibitory factor (LIF) at the termination of lactation to foster the involution of the mammary gland, thus enabling re-structuring to a state similar to prepregnacy (Chapman et al., 2002; Schere-Levy et al., 2003). When the expression of STAT3 is reduced by the interference of RNA in the breast cancer cell lines, the development of tumours in mice is inhibited. This therefore supports the significance of STAT3 in the pathogenesis of breast cancer (Ling and Arlinghaus, 2005). Hence, STATs 3 and 5 have an essential function in mammary development and breast cancer.

STATs in Cancer

Cancer is as a result of the sedition of the usual processes that offer cues for cells to develop, thrive, or segregate.  Irrespective of the main molecular defect, the effects of aberrant activation of transcription factors which cause variations in gene expression. The Signal Transducer and Activator of Transcription (STAT) family is often activated in cancer. The activation takes place due to the phosphorylation on a tyrosine. Additionally, abnormal activation of an early kinase which is inhibited by the loss of negative regulator can lead to activation of STATs in cancer (Sutherland, Lindeman, and Visvader, 2007). Most of the upstream kinases can activate STATs, however, the frequent one is the Jak family of kinases, which interrelate with different cell surface receptors.

STAT3 is activated by 70% in breast tumour constitutively, this is due to STAT3 in cancer being oncogenic and it is also recognized in all stages of breast cancer. Recent studies show that STAT3 found in mitochondria promotes the survival of breast cancer cells when it’s phosphorylated, only due to the effect of mitochondria function (Gough et al., 2009).

STAT5 expresses itself in the form of STAT5a and STAT5b, but due to the overlap of their roles, they are classified together as STAT5. Wakao, Gouilleux, and Groner (1994) found out that STAT5 fostered terminal differentiation and survival to the cell. It is also activated in the mammary gland by prolactin in cytokine, and this takes place during the last stages of pregnancy through lactation. Tweardy and Chang (2011) found out that STAT5 and prolactin to be strong barriers to the invasion and expression of cancer cell once it is activated in mesenchyal.

STAT3 and STAT5 are generally related i.e. both possess different and divergent functions in normal mammary development. Consequently, STAT5 and STAT3 have similar and divergent functions in breast cancer (Wakao, Gouilleux, and Groner, 1994).

Several tests were carried out to show the effectiveness of both transcription factors in breast cancer such as immunohistochemistry, gene expression array analysis, immunoblots, viability assay, among others. The examination of STAT3 in breast cancer was tested using breast cancer cell lines in which a panel of fourteen breast cancer cell lines were used.

STAT5 in breast cancer has also been examined by several authors. The co-activation of STAT5 and STAT3 in breast cancer were ascertained through assessing the functions of STAT3 and STAT5 in primary breast cancer. Immunohistochemistry was used to the tyrosine phosphorylated type of each protein. However, this method is limited because the small number of tumours that express STAT5 only reduces the chances of examining the clinical features of these tumours. On the other hand, the immunohistochemistry method only allows the comparison of the features of the tumours with the activation of STAT3 only against those showing activation of both STAT3 and STAT5. This is because the cancers with activation of STAT5 and STAT3 will most probably be highly differentiated grade I tumours, while STAT3 activated tumours are likely to be less differentiated grade III. This is an indication that tumours possessing both STATs have higher prognostic characteristics than those which have the activation of STAT3 only. Making use of these gene signatures, Van de Vijver et al. (2009) assessed survival in a group 295 patients suffering from primary breast cancer.

Immunohistochemistry method was also used to determine the phosphorylated STAT5 and its specificity to a certain antibody, the T-47D cells that were not treated with prolactin showed no staining compared to the cell that was treated with prolactin, which showed 100% nuclear staining (Walker et al., 2009).

The dangers of this research is high due to the usage of prolactin in their research. Prolactin is a lactogenic hormone and in the article it states that it was used to stimulate T-47D cell with prolactin while the tissue was being stained. The toxicity of prolactin is high as it increases proliferation in cell and most importantly serum prolactin increases the danger of contracting breast cancer in both premenopausal and postmenopausal in women, proven by a research conducted by Tworoger, Sluss, and Hankinson (2006).

 The specificity and reproducibility was tested by using immunohistochemistry as the antibody (phosphor-STAT5) could detect phosphorylated STAT5. Then a breast cancer tissue was stained using microarray. The antibody used was specific to phosphorylated STAT5 and STAT3 then the nuclear staining was analysed (Walker et al., 2009). Additionally, the activation of STAT3 can be improved by using STAT5 activation in breast cancer, as it moderates the effect of STAT3 (Walker et al., 2009). The article deliberates the possibility that STAT5 activation modified gene expression in human tumours.

The side effect of STAT3 once it’s activated is that it can lead to resistance to radiation and chemotherapy due to up regulation of pro-survival gene (Gritsko et al., 2006). Hence the reason why it cannot be treated alone in breast cancer as it needs co-activation with STAT5 to reduce the side effect, as it can affect the cell that consists of constitutively active STAT3 to chemotherapeutic agents.

Lavnilovitch, Groner, and Barash (2002) carried out a study using mice to show that overexpression and forced activation of STAT5 in mammary gland increases cellular proliferation, promotes differentiation, and slows apoptosis. The authors argue that signal transducer and activator of transcription 5(STAT5) has both positive and negative effect in murine models, the side effect is, STAT5 has proven to enhance mammary tumour in mice, and it can also increase the development of mammary carcinoma in mice that show constitutively activation of STAT5. The significant effect of STAT5 is the loss of the transcription factor (STAT5), which can delay tumour formation (Lavnilovitch, Groner, and Barash, 2002). Furthermore, mice that have deficiency in STAT5a are safe from mammary tumours that are induced by altering growth factor alpha. Ultimately STAT5 is the key factor in both normal and neoplastic mammary function.

A triple negative breast cancer cell line MDA-MB-468 was also used by the authors to address the issue in which the patients with tumours expressive of the activation of both STATs have improved prognosis compared to those with tumour of STAT3 activation only. The triple negative breast cancer cell line MDA-MB-468 contained constitutively active STAT3A, which were steadily moved with a constitutively active form of STAT5 (STAT5a1*6). The dual cell lines had only STAT3 activation, or the both the activation of STATs.

The hypothesis that inherent propensity of cells to go through apoptosis that is chemotherapy induced was improved when the STAT5 was activated alongside STAT3 was tested by measuring the apoptotic priming of cells using technique of BH3 profiling (Ryan, Brunelle, Letai, 2010; Ni Chonghaile et al., 2011). The authors also measured the mitochondria’s ability of depolarization in the existence of pro-apoptotic peptides.

The study by Walker et al. (2009) could be conducted better and provide more reliable results by carrying out an in vivo experiment on mouse, because a mouse has several likeness to human beings, in terms of genetics, anatomy and physiology. In this way, there is evidence to the hypothesis and the research will show improved outcomes due to the fact that a mouse genome is very similar to the human’s genome. The benefit of in vivo testing is that equally valid outcomes can be generated and thus the researchers can decide on whether the drug is safe to be tried on human beings. The experiments conducted using animals do eradicate some important drugs as either unproductive or extremely harmful for human use. Once a drug has successfully been tested on animals, it can then be tested on a sample of human beings before being applied on large scale clinical trials.

Additionally, a more significant way of obtaining better results is by using specific patients with suffering from breast cancer, who voluntarily wants to help contribute towards finding a therapeutic for breast cancer. In this way, the length of treatment will be known and how much dose is needed to realise an effect, and most importantly improve the outcomes by finding out what went wrong by conducting a better research and learning from the side effect. Another way to improve the result is using an Elisa test to produce better specificity and reproducibility. Also, another critic is that Walker, Xiang, & Frank (2014) have carried out several methodologies which can be costly to the pharmaceutical company because if they begin testing their research in vivo studying, it will require a lot of investment and the methods of techniques used will be very expensive.

To distinguish the difference between the two factors, a test to analyse the gene regulation was carried out between the two significant transducer activator of transcription (STAT3 and STAT5), also analysed the phenotype of mammary carcinoma cells. The co-activation between the two STATs have played a significant effect on the sensitivity and proliferation, based on the findings of the authors, which show that STATs reduced proliferation and improved sensitivity to the chemotherapeutic drug namely paclitaxel and vinorelbine, in contrast to STAT3 alone or STAT5 alone. Overall, it was found out that STAT3 and STAT5 regulate genes fundamental to cellular function. Additionally, these transcription factors have different and contrasting function in the development of mammary cells.

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