Activation of fibronectin/PI-3K/Akt2 leads to chemoresistance to docetaxel by regulating survivin protein expression in ovarian and breast cancer cells

Abstract

The purpose of this study was to investigate the possible role of PI-3K/Akt2 pathway in docetaxel-induced apoptosis. Here we showed that transfection of full-length Akt2 into breast and ovarian cancer cells could provoke Akt phosphorylation and induce an enhanced resistance to docetaxel. FN adhesion promoted Akt phosphorylation in highly metastatic cancer cells A2780 and MDAMB231, and further brought on significant protection for tumor cells against docetaxel-induced apoptosis. Inhibition of Akt2 activity by co-transfection with two shRNA vectors targeting the same Akt2 mRNA or simply by administration with PI 3-Kinase inhibitor Ly294002 counteracted the ability of FN to protect cells from undergoing apoptosis induced by docetaxel. We further showed that Akt2 activation protected against docetaxel-induced apoptosis by regulating survivin levels in a PI 3-Kinase-dependent manner. We conclude that FN/PI-3K/Akt2 pathway might play an important role in inducing resistance to docetaxel in breast and ovarian cancer cells. Our results therefore indicate that the activation of Akt2, promoted by FN attachment, might be critical in determining whether cells survive or undergo apoptosis. Targeting the PI-3K/Akt2 pathway might be a promising strategy for enhancing sensitivity to docetaxel in breast or ovarian cancer.

Introduction

Although chemotherapy remains a major treatment modality for human breast and ovarian cancer, chemoresistance is a clinical problem that severely limits treatment success. One of the important factors contributing to the failure of chemotherapy in patients with breast or ovarian cancer is that the cancer cells become resistant to drug-induced apoptosis. Docetaxel, a microtubule-directed drug which exert its cytotoxic effect via the induction of apoptosis, is a first-line chemotherapeutic agent in the treatment of ovarian and breast cancer. While it is generally acknowledged that the resistance of cancer cells to conventional chemotherapy is affected by multiple factors, several gene products that regulate apoptosis, i.e., PI-3K, Akt, and survivin, are frequently altered in human ovarian and breast cancer [1], [2], the most common malignancies in women. These observations suggest that inhibition of apoptosis by above-mentioned survival proteins is a key step in the development of chemoresistance.

Traditionally, drug resistance mechanisms have been identified and functionally characterized in unicellular models [3]. Implicit in these unicellular models is the lack of consideration of host–tumor cell interactions that may participate in the emergence of the drug-resistant phenotype. Thus, we suggest that in order to identify clinically relevant drug-resistant targets, new models must be developed to allow for reconstitution of host–tumor cell interactions. Several investigators have developed an adhesion-mediated drug resistance (CAM-DR) model to investigate adhesion of hematopoietic cell lineages to one specific component of the extracellular matrix, fibronectin (FN), and have shown that extracellular matrix (ECM) proteins and integrins, their cognate cell surface receptors, mediate cell adhesion while also engendering signals that participate in cell survival and resistance to cytotoxic agents [3], [4], [5].

The effective molecules involved in integrin-dependent cell survival have not been completely elucidated. Integrin-mediated adhesion to ECM stimulates the production of PI (3, 4) P2 and PI (3, 4, 5) P3, the association of the p85 PI 3-Kinase subunit with focal adhesion kinase and Akt activation [6], [7]. In turn, active Akt interferes with the apoptotic machinery by phosphorylating and thus sequestering the proapoptotic Bcl-2 family protein BAD, by inactivating members of the forkhead family of transcription factors [8]. Akt, a serine–threonine protein kinase regulated by the intracellular levels of phospholipids, is one of the central players in oncogenesis. Three member of this family, including Akt1, Akt2, and Akt3, have been identified so far [9], [10]. Among Akt family members, overexpression of Akt2 was found in 10–20% of human ovarian and breast cancers [11], [12], and increased kinase activity of Akt2 was found in approximately 30% of ovarian cancer specimens and 40% of breast cancer specimens [1], [12].

Survivin, a member of the inhibitor of apoptosis (IAP) family of proteins, is expressed during embryonic and fetal development, but is undetectable in most normal adult tissues [13]. It has been shown that survivin is required to maintain a critical anti-apoptotic threshold in prostate cancer cells, while integrin signaling has been identified as a crucial survival pathway against death receptor-mediated apoptosis via Akt pathway [14]. Whereas Akt2 and survivin are believed to be important cell survival factors in human cancer cells, if and how they interact to confer resistance to chemotherapy is unknown.

Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) has been described by Elbashir et al. [15]. Recently, a number of groups reported a solution based on transcription of short hairpin RNAs (shRNAs) by RNA polymerase III (pol III) [25], [26]. The hairpins of these short RNAs are processed to generate siRNAs and induce gene silencing. Transfection of plasmids with pol III promoters driving hairpin RNAs can eliminate the expression of a target gene. However, some siRNAs have ‘off-target’ effects (interfering with the expression or function of other genes or proteins), which are often the result of partial homology to other transcripts. To avoid ‘off-target’ effects, stringent design rules were used in gene silencing experiments in addition to published siRNA design guidelines. Furthermore, two or multiple siRNAs directed against different regions of the same target were suggested [16], [17]. In this study, to avoid ‘off-target’ effects, two independent 19-nucleotide [11] sequences targeting Akt were designed, and were cloned into two-shRNA expression vectors, respectively.

In the present study, we focused on the role of PI-3K/Akt2 signal pathway activation in acquired resistance to docetaxel in ovarian and breast cancer cells as well as the relationships among FN, Akt2, and survivin in chemoresistance. CAM-DR model was used in vitro instead of traditional unicellular models. We established two shRNA expression vectors directed against different regions of the same target Akt2 for assessing Akt2 gene function. Here we report that activation of the FN/PI-3K/Akt2 pathway led to docetaxel resistance in highly metastatic ovarian and breast cancer cells. Our results further demonstrate a novel mechanism by which Akt2 regulates apoptosis and a possible role for Akt2 activation, promoted by FN attachment in the protection of survivin against docetaxel-induced down-regulation.