The PI K PTEN Akt

The PI3K/PTEN/Akt/mTORC1/GSK-3 pathway also plays key roles in metabolism, obesity, diabetes and cardiovascular diseases (, , , , ). Sometimes such clinical problems such as diabetes, obesity and metabolic disorders are interlinked with cancer. Clearly further elucidation of the roles of GSK-3 could potentially alleviate treatment of various diseases and improve therapeutic outcomes. Conflicts of interest
Acknowledgements JAM was supported in part by a grant from: East Carolina University Grant (#111104). LC was supported in part by grants from: Intesa San Paolo Foundation.
Introduction Breast cancer is the leading cause of cancer deaths in women worldwide [1]. Despite enormous advances in early diagnosis and surgical treatment of breast cancer, as well as improvements in neoadjuvant and adjuvant therapy, approximately 30% of patients with breast cancer will develop incurable metastatic disease [2]. Treatment with conventional chemotherapeutic drugs has had little impact on metastatic disease progression. Metastatic breast cancer remains an incurable disease with median survival times ranging from one to four years depending on the subtype [2], [3], and thus represents a significant unmet medical need. The identification of new targeted therapeutic agents is urgently needed. Glycogen Synthase Kinase-3 (GSK-3), a serine/threonine protein kinase, was initially described as a key enzyme involved in glycogen metabolism [4], [5] but is now recognized as a regulator of diverse cellular functions [6]. GSK-3 phosphorylates and thereby regulates the activity of many metabolic, signaling, and structural proteins [6]. There are two highly homologous forms of GSK-3 in mammals, GSK-3α and GSK-3β [6]. Historically, GSK-3β has been thought of as a potential tumor suppressor due to its ability to phosphorylate and thereby target pro-oncogenic molecules including c-Jun [7], c-Myc [8], cyclin D1 [9] and β-catenin [10] for ubiquitin-dependent proteosomal degradation. However, recent reports have suggested that GSK-3β is a positive regulator of cancer cell proliferation and survival [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22] providing further support for GSK-3β as a therapeutic target in cancer. Previously, we identified GSK-3β as a novel therapeutic target in human leukemia, pancreatic, colon, apexbio dilution and renal cancer [11], [14], [15], [16], [17]. In human breast carcinoma, it has been shown that overexpression of GSK-3β was associated with several indicators of poor prognosis and breast cancer patients with GSK-3β expression in the highest quartile (246 of 1686 cases) had a 2.7 and 1.7-fold increased risk of distant relapse 5 and 10 years after tumor resection, respectively [23]. A recent study demonstrated that GSK-3β knockdown significantly inhibited breast cancer cell proliferation whereas GSK-3α knockdown had only a minor effect in four breast cancer cell lines further credentialing GSK-3β as a viable therapeutic target for the treatment of breast cancer [24]. We recently identified two lead ATP-competitive GSK-3β inhibitors, 9-ING-41 and 9-ING-87, based on the potency of their antiproliferative activity against pancreatic and ovarian cancer cells in vitro as well as supportive ADMET and PK studies [13], [25]. Here, we describe the anti-tumor activity of these novel inhibitors of GSK-3 in both breast cancer cell lines and in novel patient-derived xenograft (PDX) models of metastatic chemorefractory breast cancer. We conclude that the inhibition of GSK-3 is a promising therapeutic approach to overcome chemoresistance in metastatic breast cancer.
Materials and methods
Results
Discussion Recently, GSK-3β has been credentialed as a potential therapeutic target in human breast cancer [23], [24]. GSK-3β knockdown significantly inhibited breast cancer cell proliferation whereas GSK-3α knockdown had only a minor effect in four breast cancer cell lines, providing further support for GSK-3βas a breast cancer therapeutic target [24]. In a recently published study of 1686 cases of breast cancer, overexpression of GSK-3β was associated with several indicators of poor prognosis including lymph node metastasis, increased tumor size, high pathological grade, ER-negative disease, PR-negative disease, increased proliferation (measured by Ki-67) and HER2 overexpression [23]. Moreover, breast cancer patients with GSK-3β expression in the highest quartile (246 of 1686 cases) had a 2.7 and 1.7-fold increased risk of distant relapse 5 and 10 years after tumor resection, respectively [23]. In the present study, we describe a novel GSK-3 inhibitor 9-ING-41 which shows robust antitumor activity in vitro and in vivo and possesses drug-like properties [13], [25], [32]. Our in vitro results demonstrate that 9-ING-41 is a more potent inhibitor of breast cancer cell growth than other available GSK-3 inhibitors including the clinical stage compound LY2090314. We demonstrate that inhibition of GSK-3 by 9-ING-41 decreases the survival of breast cancer cells in vitro, consistent with previously published studies in other tumor cell types [11], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [25], [32], [33], [34], [35], [36]. Our previous studies in leukemic cells showed that the inhibition of GSK-3 using a toolkit inhibitor suppressed NF-κB transcriptional activity and decreased the expression of the antiapoptotic proteins (XIAP, Bcl-2), leading to enhaced cancer cell apoptosis [15]. Given that NF-κB-mediated chemoresistance has been shown to be a major driver of breast cancer progression [37], [38], we examined inhibition of GSK-3 by 9-ING-41 in breast cancer cells in vitro and found it decreased the expression of anti-apoptotic molecules NF-κB target genes Bcl-2 and XIAP inducing apoptosis. We then tested whether 9-ING-41 could enhance the response of the chemoresistant metastatic breast cancer cell lines to the chemotherapeutic drug CPT-11 in vitro. Chemoresistance of cancer cells in vitro is defined by many factors including drug concentration and duration of exposure in cell culture. Because continuous exposure (72–96 hours) of breast cancer cells to either 9-ING-41 or a chemotherapeutic drug significantly suppresses cell viability and does not mimic tumor exposure to a drug in vivo, we carried out short term (3 hours) exposures of breast cancer cell lines using a minimal effective inhibitory concentration of 9-ING-41 in combination with a chemotherapeutic drug followed by washout with fresh drug-free cell culture media and then allowed the cells to grow for 72 hours. Under these conditions, we found that 9-ING-41 potentiates the antitumor effects of CPT-11 on breast cancer cell growth. Our in vitro results suggested that inhibition of GSK-3 by 9-ING-41 sensitized metastatic breast cancer cells to CPT-11 chemotherapy, a potent drug that has otherwise limited clinical activity in this indication.