Targeted Treatment Of Breast Cancer And Acute Myeloid Leukemia

Background:Amplification and/or overexpression of erbB2occur in approximately25%of invasive breast cancer and are significantly associated with a worse prognosis for breast cancer patients. The erbB3receptor plays a critical role in erbB2-overexpressing breast cancer, and its elevated expression induces resistance to therapeutic agents, including trastuzumab. Our recent studies indicate that erbB3interacts with both erbB2and IGF-1receptor to form a heterotrimeric complex in trastuzumab-resistant breast cancer cells. Herein, we investigate the antitumor activity of MM-121, a humanized anti-erbB3antibody, against erbB2-overexpressing breast cancer cells resistant to trastuzumab in vitro and in vivo.Methods:Cell Titer AQueous Non-Radioactive Cell Proliferation Assays (MTS) were used to determine cell proliferation. Cell cycle progression was examined by flow cytometric analyses. Western blot analyses were performed to determine expression and activation of proteins upon treatments. Tumor xenograft model were established by inoculation of the trastuzumab-resistant BT474-HR20cells into nude mice. The tumor-bearing mice were treated with trastuzumab and/or MM-121/SAR256212via i.p. injection to determine the Abs’ antitumor activity. Immunohistochemical analyses were carried out to study the Abs’inhibitory effects on tumor cell proliferation and induction of apoptosis in vivo.Results:Cell proliferation assays showed that MM-121significantly enhanced trastuzumab-induced growth inhibition in both sensitive and resistant breast cancer cells. MM-121in combination with trastuzumab mainly resulted in a dramatic reduction of phosphorylated erbB3(P-erbB3) and the downstream signaling P-Akt. MM-121combined with trastuzumab did not lead to apoptosis in trastuzumab-resistant cells in in vitro cell culture condition, rather induced cell cycle G1arrest which was associated with a slight decrease of E2F-1and upregulation of p27kip1. Interestingly, in the mouse tumor xenografts model established from trastuzumab-resistant cells, MM-121in combination with trastuzumab as compared to either agent alone significantly inhibited tumor growth correlated with a significant reduction of Ki-67staining and increase of cleaved caspase-3in the tumor tissues. These data suggest that the combination of MM-121and trastuzumab not only inhibits tumor cell proliferation, but also promotes the resistant cells undergoing apoptosis in vivo.Conclusions:We demonstrate that MM-121exhibits potent antitumor activity when combined with trastuzumab in vitro and in vivo. MM-121may be added into the treatment regimens for breast cancer patients with erbB2overexpression and resistance to trastuzumab. Background:Survivin (coding gene:BIRC5) is the smallest mammalian member of IAPs (inhibitor of apoptosis proteins) family, a dual functional protein acting as a critical apoptosis inhibitor and key cell cycle regulator. Survivin is usually expressed in embryonic tissues during development and undetectable in most terminally differentiated tissues. Numerous studies demonstrate that survivin is selectively upregulated in almost all types of human malignancies and its overexpression positively correlates with poor prognosis, tumor recurrence, and therapeutic resistance. Survivin plays a vital role in normal hematopoiesis;however, overexpression of survivin is reported in different kinds of hematological malignancies correlating with inferior clinical outcome. Studies with acute myeloid leukemia (AML) patients reveal the correlation between higher levels of survivin and worse prognosis. There are few studies about targeting survivin in treating AML. It is still unclear how AML cells’chemosensitivity change upon downregulation of survivin. Thus, we use survivin specific shRNA to knockdown its expression in AML cells, evaluating how AML cells react and how their chemo-sensitivity change upon survivin downregulation. We also investigate functions of the small molecular survivin inhibitor YM155on AML cells, used as single reagent or combined with other chemotherapeutics. We want to provide rationale for using survivin as a target in AML treatment.Methods:Traditional PCR and real-time PCR were used to determine the mRNA levels in AML cell lines. Western blot analyses were performed to determine the protein levels in AML cell lines and the expression and activation of proteins upon treatments. Cell death was quantified by a specific apoptotic ELISA (enzyme linked immunosorbent assay). Flow cytometric analyses were used to determine cell cycle change upon survivin knockdown or YM155treatment. Cell Titer AQueous Non-Radioactive Cell Proliferation Assays (MTS) were carried out to investigate cell proliferation change upon survivin knockdown or YM155and chemotherapeutics single/combined treatments.Result:Kasumi-1and HL-60cells have the highest expression of survivin among all the tested AML cells. Specifically knocking down the expression of survivin in Kasumi-1and HL-60cells resulted in:inhibition of cell proliferation; cell cycle G2/M phase arrest; upregulation of DNA damage and apoptotic related proteins; increased histone associated DNA fragmentation. Knocking down survivin with specific shRNAs also increased the chemosensitivity of AML cells. Comparing with non-target control group, chemotherapeutics induced more apoptotic and DNA damage related proteins in survivin silenced groups. Small inhibitor YM155trigged does and time dependent reduction of survivin in Kasumi-1and HL-60cells. Cells death caused by YM155was also dependent on dose and time. The combination effect of YM155and chemotherapeutics can be synergetic or antagonistic, depending on the drug used for combination and the type of cell being treated.Conclusion:Our data demonstrated survivin is important for the maintenance and proliferation of AML cells. Knocking down survivin in AML cells enhanced their chemosensitivities. YM155induced survivin downregulation and cell death of AML cell in a dose and time dependent manner. The combination effect of YM155and chemotherapeutics can be synergetic or antagonistic, depending on the drug used for combination and the type of cell being treated. Our findings provide strategies for targeting survivin in treating AML, giving rationale for further in vivo and clinical studies.