Translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613.

STUDY RATIONALE AND OBJECTIVES: Via genetic alterations, malignant transformation and proliferation are associated with extensive alterations of mitochondrial energy metabolism of tumor cells. Thus, inhibition of the altered form of mitochondrial energy metabolism of tumor cells may be an effective therapy for cancers. This study performed translational assessment of mitochondrial dysfunction of pancreatic cancer from in vitro gene microarray and animal efficacy studies, to early clinical studies, via the novel tumor-specific anti-mitochondrial agent, CPI-613. METHODS: The gene profiles of BxPC-3 human pancreatic tumor cells and non-transformed NIH-3T3 mouse fibroblast cells (negative control), after CPI-613 or sham treatment, were assessed and compared using microarray technique. The anti-cancer efficacies of CPI-613 and Gemcitabine were assessed and compared in mice with xenograft from inoculation of BxPC-3 human pancreatic tumor cells, based on the degree of tumor growth inhibition and prolongation of survival when compared to vehicle treatment. The anti-cancer activities, according to overall survival (OS), of CPI-613 alone and in combination with Gemcitabine were assessed in patients with Stage IV pancreatic cancer. RESULTS: Microarray studies indicated that CPI-613 down-regulated the expression of Cyclin D3, E1, E2, F, A2, B1 and CDK2 genes of BxPC-3 pancreatic cancer cells but not non-transformed NIH-3T3 mouse fibroblast cells (negative control). In mice with pancreatic carcinoma xenografts, four weekly intraperitoneal injections of either CPI-613 (25 mg/kg/administration) or Gemcitabine (50 mg/kg/administration) inhibited tumor growth and prolonged survival when compared to vehicle treatment. The degree of tumor growth inhibition was ~2×, and prolongation of survival was ~4×, greater with CPI-613 treatment than with Gemcitabine treatment. In patients with Stage IV advanced pancreatic cancer, CPI-613 at 420-1,300 mg/m(2), given twice weekly for three weeks followed by a week of rest (i.e., 3-week-on-1-week-off) as monotherapy, provided median OS of 15 months in three patients. CPI-613 at 150-320 mg/m(2) given twice weekly on the 3-week-on-1-week-off dosing schedule, coinciding with Gemcitabine (1,000 mg/m(2)) given once weekly on the 3-week-on-1-week-off dosing schedule, provided median OS of 17.8 months in four patients. These median OS values from CPI-613 monotherapy and CPI-613 + Gemcitabine treatment tend to be longer than those in patients treated with Abraxane + Gemcitabine combination or FOLFININOX (median OS ~12 months). CONCLUSIONS: The dysfunctional mitochondria of pancreatic cancer cells was translationable from in vitro gene alteration and animal tumor model studies to patients with advanced Stage IV pancreatic cancer, as reflected by the anti-cancer activities of the tumor-specific anti-mitochondrial agent, CPI-613, in these studies.

Leptin-regulated gene expression in MCF-7 breast cancer cells: mechanistic insights into leptin-regulated mammary tumor growth and progression.

Obesity is a recently established risk factor for breast cancer incidence and mortality. A characteristic of obesity is elevated circulating levels of adipocyte-derived hormone leptin. Evidence indicates that leptin plays an important role in mammary tumor formation; however, the mechanisms involved are poorly understood. Toward better defining the role of leptin in breast cancer, we describe the identification of leptin-regulated genes in hormone-responsive Michigan Cancer Foundation-7 (MCF-7) human breast cancer cells using a microarray system. More than 64 leptin-regulated genes were identified including those for growth factors, cell cycle regulators, extracellular matrix (ECM) proteins, and genes associated with metastasis. Cell cycle genes up-regulated by leptin include cyclins D and G, cyclin-dependent kinase 2, p21, p27, and p16. Leptin suppressed the expression of transforming growth factor-beta , a cell cycle suppressor. Determining the significance of this effect, treatment of MCF-7 cells with TGFB1 abrogated leptin-stimulated proliferation. Leptin up-regulated the expression of connective tissue growth factor, villin 2, and basigin, factors that are associated with ECM and are known to impact tumor growth. Finally, leptin induced the expression of anti-apoptotic genes BCL2 and survivin, and reduced the expression of apoptotic genes. The effect of leptin on MCF-7 survival was evaluated via TUNEL assay and demonstrated a sixfold reduction in apoptosis in leptin-treated cells, compared with controls. These data suggest leptin promotes mammary tumor growth through multiple mechanisms, including regulating the cell cycle, apoptosis, and by modulating the extracellular environment. The identification of leptin-regulated genes begins to provide mechanistic links into the relationship between obesity and breast cancer incidence and morbidity.

Identification of proteins secreted from leptin stimulated MCF-7 breast cancer cells: a dual proteomic approach.

Leptin is an adipocyte-derived hormone that regulates energy expenditure and food intake. A significant role for leptin in breast cancer has also been indicated by the resistance of leptin knockout mice in development of mammary tumors. In vitro, leptin induces proliferation of MCF-7 cells by activating cellular signaling pathways (1, 11, 12, 16, 17, 56). As leptin is emerging as an important factor for tumor growth, and hormones can exert their actions via autocrine/paracrine mechanisms, we hypothesized leptin may act by regulating epithelial-derived proteins. To test this hypothesis, leptin-regulated proteins secreted from MCF-7 mammary tumor cells were identified using proteomics techniques. Treatment of MCF-7 cells with 500 ng/ml leptin for 24 hours resulted in a 40% increase in cell number and a 5-fold increase in protein secretion as compared to controls. Establishing the significance of leptin-induced secreted factors, the addition of conditioned media from leptin-treated MCF-7 cells to synchronized MCF-7 cells resulted in 40% increase in cell number. Identification of leptin-regulated secreted proteins was done by 2D gel electrophoresis coupled with MALDI-TOF mass spectrometry. Proteins identified using Pro Found software and NCBI database included KF10 Collagen Precursor, Serologically Defined Breast Cancer Antigen NY-BR-62 and Cortactin Isoform a. A Human Cytokine Antibody Array system was used to identify low abundant proteins in the media of control and 500 ng/ml leptin-stimulated MCF-7 cells. In leptin treated cells, levels of FGF-9 were increased while IGFBP-3 and TGF-beta3 levels were decreased. Many previous studies have focused on the regulation of distinct cellular proteins by leptin during mammary tumor cell proliferation. However, ours is the first study to identify leptin-regulated secreted proteins, many of which are known to play important roles in cancer. Our data support that leptin can influence mammary tumor growth and progression through regulation of autocrine/paracrine factors and by modulating the extracellular matrix composition.