Role of Prolactin and Prolactin Receptor Signaling in Colorectal Tumorigenesis

Abstract

Hormones are critical regulatory factors produced by the body to regulate diverse physiological activities such as energy homeostasis, growth and differentiation of a diverse array of tissues, sexual maturation and development of secondary sexual characters. Cytokines are hormones, which predominantly regulate growth, proliferation differentiation, immunomodulation and tumor progression. They act in endocrine, paracrine or autocrine manner, and elicit their action by binding to a cell surface receptor and activating diverse intracellular signaling cascades. Prolactin (PRL) is a peptide hormone, encoded by the PRL gene located on chromosome 6 in humans. This gene is under the control of two independent promoters, the pituitary promoter which regulates its expression from the lactotroph cells of the anterior pituitary and an extrapituitary promoter which regulates its expression from extra-pituitary tissues such as endometrium, placenta, breast and a variety of tumors. PRL in humans binds specifically to PRL receptor (PRLR), to cause intracellular changes modulated mainly via the JAK-STAT or JAK-STAT-ERK pathways. Historically, PRL has been studied as an endocrine hormone that regulates lactation during pregnancy. However, the identification of extra-pituitary PRL and the complex clinical consequences of hyperprolactemia have prompted investigators to reevaluate its role in regulating other physiological aspects. Studies from several groups over the last few decades have shown that PRL can regulate a spectrum of functions ranging from behavior to immune responses to tumorigenesis. Cancer is a growth disorder which was mentioned as early as 460-370 BC, by Hippocrates who coined the term "cancer" based on its appearance post-surgery. Throughout history, cancer has been the cause of severe physical and emotional suffering and death in humans and animals alike. Cancer has an exceptional capability to take over body's normal physiology, modulate it and uses it for its own growth and to overcome anti-cancer treatment. Over the last several decades, research efforts from several laboratories have helped gain a better understanding of the molecular mechanisms that regulate cancer initiation, development and progression. These studies have also provided new insights for identifying and developing new pharmaceutical compounds to target tumor cells. Colorectal cancer (CRC) is the third leading cause of cancer related death in United States. Worldwide, up to 5% of all reported cancer cases are due to CRC, with 60% of them being diagnosed in industrially developed or developing countries. CRC is caused by genetic and environmental factors. Environmental factors ranging from changing dietary habits to environmental toxins are associated with the development of CRC. Germline mutations in APC, TP53 and DNA mismatch repair genes gives rise to familial inheritable form of CRC and contribute to nearly 35% of the registered CRC cases. This dissertation outlines the expression pattern of PRLR and the cellular mechanism(s) which are regulated or activated by PRL- PRLR signaling and the contribution of this signaling towards pathogenesis of CRC. We have determined that CRC cells treated with recombinant human PRL show a time- and dose-dependent phosphorylation of JAK2, STAT3 and ERK1/2 proteins. Previous studies have demonstrated that breast cancer cells treated with PRL show a rapid induction of STAT5 phosphorylation. However, in our studies, we found that colon cancer cells treated with PRL show an induction of STAT3 phosphorylation. This may be in part due to low basal level of STAT5 in colon cancer cells. In addition, PRL treatment does not lead to increase in proliferation, falling in line with earlier observations, that STAT3 is not a proliferation promoting factor. Pre-incubating CRC cells with AG490 and PD98059 which are established JAK2 and ERK1/2 inhibitors prior to PRL treatment led to a complete abrogation of respective phosphorylation, suggesting that the observed activation of JAK2 and ERK1/2 is indeed induced by PRL in CRC cells. PRL treatment induces spheroid formation, a hall mark of cancer stem cells and does so, by activating Notch signaling. The Notch signaling pathway is critical in maintaining cancer stem cell populations both in vitro and in vivo. PRL activates Notch signaling by inducing the expression of Jagged 1(JAG1), a Notch receptor ligand. Binding of JAG1 to Notch receptor induces conformation changes in the receptor, leading to its cleavage and translocation of the cleaved intracellular domain (NICD) into the nucleus where it activates expression of Notch responsive genes. PRL treatment induces a time dependent increase in Notch cleavage. In addition, an increase in expression of Hes1 and Hey1, established Notch target genes, clearly implicate PRL treatment in activation of Notch signaling in colon cancer cells. In addition, the treatment induced expression of established colon cancer stem cell marker proteins such as LGR5, DCLK1 and CD44 suggests that PRL contributes towards modulating colon cancer stem cell population. Pretreating CRC cells with AG490 and PD98059, leads to loss of Notch activation and decreased expression of cancer stem cell marker proteins, again implicating the role of PRL in modulating the Notch signaling, thereby playing a critical role in regulating colon cancer stem cell population. One of the critical aspects associated with human PRL signaling is its receptor specificity. Human PRL can bind only to PRLR, which is a specific PRL receptor. Our findings indicate that PRL can modulate critical aspects associated with colorectal cancer. We were interested in examining the expression pattern of PRLR in colorectal cancer patients with an aim to develop novel diagnostic tools or therapeutically target PRL signaling. Our findings clearly indicate that PRLR is expressed in normal tissues throughout the GI tract, with predominant expression in the large intestine. Additionally, PRLR expression is significantly increased in colorectal cancer biopsy samples compared to adjacent normal samples. This suggests that PRL signaling can play a critical role in colorectal cancer tumorigenesis. A couple of factors may contribute to the increase in expression of a gene: first being an increase in copy number of the gene and second being an enhanced transcription of the gene. In order to examine the existence of chromosomal variation, we analyzed TCGA data sets pertaining to expression and copy number data. Data analysis suggested the possibility of an increase in copy number of not only PRLR but also of a couple other genes located in the vicinity in some patients. Among the other patients, some had an increase in expression without any change in copy number. To identify the reason for increased expression of PRLR in these patients, we evaluated the possibility of tumor specific transcription factor binding and subsequent increase in PRLR transcription. We analyzed a 2 Kb upstream region of the PRLR promoter and identified binding sites for SREBP-1, a transcription enhancer that regulates expression of enzymes necessary for lipid metabolism and energy homeostasis. SREBP-1 is highly expressed in colorectal tumors. Studies using ChIP and RT-PCR analysis indicate that SREBP-1 is actively recruited to the PRLR enhancer region and can potentially be involved in regulating its expression. Collectively, this dissertation provides novel insights into the role of PRL in colorectal tumorigenesis. It also implicates the critical role of PRLR signaling in colorectal cancer and suggests that PRLR can be exploited as a diagnostic marker.