Mechanism of Anti-Proliferative Action of 2-[Piperidinoethoxyphenyl]-3-Phenyl-2h-Benzo(B)Pyran in Estrogen Receptor Positive- and Estrogen Receptor Negative- Human Breast Cancer Cell Lines

Abstract

Breast cancer is the second most commonly diagnosed malignancy among women with more than one million new cases diagnosed per year throughout the world [1]. Despite advances in the early detection of breast cancer and the advent of novel targeted therapies, breast cancer still remains a significant public health problem due to the involvement of multiple aberrant and redundant signaling pathways in the tumorigenesis and the development of resistance to the existing therapeutic agents. With the increasing understanding of the cellular processes involved in breast cancer at the molecular level, focus was directed towards the development of targeted therapies. The first selective and targeted therapy came when it was found that the majority of cases of breast cancer express higher levels of estrogen receptor α (ERα) [2, 3]. With the evolving understanding of the biology of ERα pathways, selective ER modulators (SERMs) were developed that represented the first targeted endocrine therapy [4, 5]. Tamoxifen is currently the first-line endocrine agent for the treatment of ERα-positive primary and advanced breast cancers [5, 6]. However, there are some negative side-effects associated with it that include increased incidences of endometrial cancer in post menopausal women, increased blood clots and increased cataracts [7-9]. Another problem with the use of Tamoxifen is that all patients with metastatic disease and about 40% patients on adjuvant Tamoxifen therapy eventually relapse and ultimately die from this disease and many ERα-positive patients do not respond to Tamoxifen therapy at all [10, 11]. Another endocrine therapy evolved after the development of Tamoxifen resistance included pure antiestrogens like Fulvestrant [12]. However studies report that long term pure antiestrogen treatment may lead to the development of resistance to the agent [13]. In many clinical cases, the lack of response to endocrine therapy together with increased metastasis was found to be associated with overexpression of epidermal growth factor receptor (EGFR) / Herceptin-2 (HER-2) and increased crosstalk of this pathway with ERα [14, 15]. Apart from the development of resistance to endocrine therapy in ER- positive tumors, the major issue of concern has been the subgroup of patients with estrogen receptor negative breast cancer that do not respond to the endocrine therapy and have poor prognosis [16]. These ER- negative tumors are high grade tumors and are more invasive. In ER- negative tumors, overexpression of EGFR or HER-2, leading to increased growth factor signaling, is observed such that various cell survival and proliferation pathways are significantly hyper-activated as compared to ER- positive breast cancer [17, 18]. Thus, targeting the growth factor-mediated signaling has become an important therapeutic option for breast cancer treatment. Development of EGFR inhibitors began some 15-20 years ago [19, 20]. Gefitinib is a small-molecule anilinoquinazoline that reversibly inhibits EGFR tyrosine kinase autophosphorylation and inhibits downstream signaling [21, 22]. Another small molecule, Lapatinib (lapatinib ditosylate) is an orally active dual inhibitor of the tyrosine kinase domain of both EGFR and HER-2 [23, 24]. This novel investigational agent has given enthusiastic results in patients with metastatic, treatment-refractory disease [25]. It was approved in March 2007 for use in combination with Capecitabine in patients with advanced, refractory metastatic breast cancer [26, 27]. Lapatinib is also found to be useful in inflammatory breast cancer patients [28]. Recently, Lapatinib treatment in a neo-adjuvant setting has shown to decrease the number of breast cancer stem cells and in tumor biopsies as opposed to chemotherapy which led to an increase [29, 30]. Hence, Lapatinib holds the potential to become the mainstay of EGFR/HER-2 positive breast cancer therapy in future. Another recently explored target for breast cancer is Murine Double Minute 2 (Mdm2) protein and its interaction with tumor suppressor p53 [31]. Mdm2 is amplified or overexpressed in many human cancers, including breast cancer, and Mdm2 levels are associated with poor prognosis. The Mdm2 oncoprotein promotes cell survival and cell cycle progression by inhibiting the p53 tumor suppressor protein [32]. However in case of p53 mutated or deleted tumors Mdm2 exerts its effects in a p53 independent manner by interacting with protein like pRb, E2F1, p21 and Bax [33]. Designing small molecules to block the Mdm2-p53 interaction and reactivate the p53 function is a promising therapeutic strategy for the treatment of cancers retaining wild type p53. Several classes of compounds including imidazoline, benzodiazepine and spirooxindoles have been synthesized in the past few decades that are known to disrupt Mdm2-p53 interaction thus reactivating the tumor suppressive properties of p53 [34]. Recently, potent and selective small-molecule antagonists of Mdm2, Nutlins, have been identified [35]. Nutlins bind Mdm2 in the p53-binding pocket and activate the p53 pathway in human cancer cells with wild-type p53, leading to cell-cycle arrest, apoptosis, and growth inhibition of human tumor xenografts in nude mice [36, 37] Using structure-based design, Ding et al. (2005) identified several compounds with a spiro-oxindole core structure that can inhibit Mdm2-p53 binding in-vitro [38]. The most potent compounds in this class have shown anti-proliferative activity in the low micromolar range with nearly 30-fold selectivity for cells with wild type p53 over cells with mutant p53, but their biological activity has not been well characterized. The spiro-oxindole analogs MI-63 and MI-219 have shown the potential of using pharmacological activation of p53 by disrupting the Mdm2-p53 interaction as an anticancer strategy for cancers retaining wild-type p53 [39, 40]. Thus future strategy should include agents that could simultaneously target pathways involved in pathogenesis of ER-positive and ER-negative breast cancer. Novel therapeutic agents specifically targeted at the inhibition of ER as well as growth factor receptors and events within the signal transduction pathways constitute an ideal approach for the treatment of breast cancer patients. In search for better therapeutic agent, we targeted ER, growth factor receptor EGFR and Mdm-2-p53 interaction using small molecule inhibitors. To that end, we screened synthetic series consisting of benzopyran derivatives and spiro-oxindole derivatives against human breast cancer cells, evaluated the efficacy of the lead molecules and finally validated the molecular targets. The study has been divided into four chapters: Anti-tumor activity and mechanism of action of 2-[piperidinoethoxyphenyl]-3- phenyl- 2H-benzo (b) pyran (CDRI-85/287) in estrogen receptor positive breast cancer cells and xenograft model Anti-tumor activity and mechanism of action of CDRI-85/287 in estrogen receptor negative breast cancer cells and xenograft model Anti-proliferative activity of a novel series of spiro-oxindole derivatives in breast cancer cells and dissecting the mechanism of action of lead compound 5-chloro-4',5'-diphenyl-3'-(4-(2-(piperidin-1-yl) ethoxy) benzoyl) spiro [indoline-3,2'-pyrrolidin]-2-one (G613) in breast cancer cells expressing wild type p53 and in xenograft model. Deciphering the molecular mechanism of lead spiro-oxindole compound G613 in p53- mutated breast cancer cells and its anti-tumor activity in xenograft model.