Abstract
IntroductionElevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Here, we study the antitumor activity of an anti-erbB3 antibody MM-121/SAR256212 in combination with paclitaxel against erbB2-overexpressing breast cancer.MethodsCell growth assays were used to determine cell viability. Cells undergoing apoptosis were quantified by a specific apoptotic ELISA. Western blot analyses were performed to assess the protein expression and activation. Lentiviral vector containing shRNA was used to specifically knockdown Survivin. Tumor xenografts were established by inoculation of BT474-HR20 cells into nude mice. The tumor-bearing mice were treated with paclitaxel and/or MM-121/SAR256212 to determine whether the antibody (Ab) enhances paclitaxel’s antitumor activity. Immunohistochemistry was carried out to study the combinatorial effects on tumor cell proliferation and induction of apoptosis in vivo.ResultsMM-121 significantly facilitated paclitaxel-mediated anti-proliferative/anti-survival effects on SKBR3 cells transfected with a control vector or erbB3 cDNA. It specifically downregulated Survivin associated with inactivation of erbB2, erbB3, and Akt. MM-121 enhances paclitaxel-induced poly(ADP-ribose) polymerase (PARP) cleavage, activation of caspase-8 and -3, and apoptosis in both paclitaxel-sensitive and -resistant cells. Specific knockdown of Survivin in the trastuzumab-resistant BT474-HR20 cells dramatically enhanced paclitaxel-induced apoptosis, suggesting that increased Survivin caused a cross-resistance to paclitaxel. Furthermore, the studies using a tumor xenograft model-established from BT474-HR20 cells revealed that either MM-121 (10 mg/kg) or low-dose (7.5 mg/kg) paclitaxel had no effect on tumor growth, their combinations significantly inhibited tumor growth in vivo. Immunohistochemical analysis showed that the combinations of MM-121 and paclitaxel significantly reduced the cells with positive staining for Ki-67 and Survivin, and increased the cells with cleaved caspase-3.ConclusionsThe combinations of MM-121 and paclitaxel not only inhibit tumor cell proliferation, but also promote erbB2-overexpressing breast cancer cells to undergo apoptosis via downregulation of Survivin in vitro and in vivo, suggesting that inactivation of erbB3 with MM-121 enhances paclitaxel-mediated antitumor activity against erbB2-overexpressing breast cancers. Our data supports further exploration of the combinatorial regimens consisting of MM-121 and paclitaxel in breast cancer patients with erbB2-overexpressing tumors, particularly those resistant to paclitaxel.
Highlights
Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/as PI-3 K/protein kinase B (Akt)-dependent upregulation of Survivin
Western blot analyses showed that treatment of SKBR3.neo1, SKBR3.B3.1, and SKBR3.B3.2 cell lines with MM-121 reduced the levels of phosphorylated erbB3 (P-erbB3) and phosphorylated erbB2 (P-erbB2) in a time-dependent manner (Figure 1B)
Treatment with MM-121 gradually reduced the levels of phosphorylated Akt (P-Akt), but had no significant effects on phosphorylated mitogenactivated protein kinase (MAPK) (P-MAPK) (Figure 1B)
Summary
Elevated expression of erbB3 rendered erbB2-overexpressing breast cancer cells resistant to paclitaxel via PI-3 K/Akt-dependent upregulation of Survivin. It is unclear whether an erbB3-targeted therapy may abrogate erbB2-mediated paclitaxel resistance in breast cancer. Most metastatic breast cancers show expression for either EGFR or erbB2, and less often for both [7]. ErbB3 has been shown to serve as a critical co-receptor of erbB2, and its expression is a rate-limiting factor for erbB2mediated breast cancer cell survival and proliferation [13,14]. We and others have observed an elevated expression of the endogenous mouse erbB3 in the mammary tumors derived from erbB2/neu-transgenic mice [15,16]; and the increased erbB3 forms physical and functional interactions with the transgene-encoded erbB2 to promote mammary tumorigenesis [15]. The erbB2/ erbB3 heterodimer has been identified as the most potent form of all erbB receptor complexes to activate the oncogenic signaling, such as PI-3 K/protein kinase B (Akt), mitogen-activated protein kinase kinase (MEK)/mitogenactivated protein kinase (MAPK), and/or janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathways, and/or Src kinase, in breast cancers [17,18,19]
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