Fangping Zhao

Systemic Treatment with the Antidiabetic Drug Metformin Selectively Impairs p53-Deficient Tumor Cell Growth

The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53(+/+) and HCT116 p53(-/-). Treatment with metformin selectively suppressed the tumor growth of HCT116 p53(-/-) xenografts. Following treatment with metformin, we detected increased apoptosis in p53(-/-) tumor sections and an enhanced susceptibility of p53(-/-) cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53(-/-) tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53(+/+) cells, but not HCT116 p53(-/-) cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid beta-oxidation in p53(+/+) MEFs, but not in p53(-/-) MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53(-/-) cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin.

Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation.

Production of a red blood cells hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.

ATP citrate lyase is an important component of cell growth and transformation

Cell proliferation requires a constant supply of lipids and lipid precursors to fuel membrane biogenesis and protein modification. Cytokine stimulation of hematopoietic cells directly stimulates glucose utilization in excess of bioenergetic demand, resulting in a shift from oxidative to glycolytic metabolism. A potential benefit of this form of metabolism is the channeling of glucose into biosynthetic pathways. Here we report that glucose supports de novo lipid synthesis in growing hematopoietic cells in a manner regulated by cytokine availability and the PI3K/Akt signaling pathway. The net conversion of glucose to lipid is dependent on the ability of cells to produce cytosolic acetyl CoA from mitochondria-derived citrate through the action of ATP citrate lyase (ACL). Stable knockdown of ACL leads to a significant impairment of glucose-dependent lipid synthesis and an elevation of mitochondrial membrane potential. Cells with ACL knockdown display decreased cytokine-stimulated cell proliferation. In contrast, these cells resist cell death induced by either cytokine or glucose withdrawal. However, ACL knockdown significantly impairs Akt-mediated tumorigenesis in vivo. These data suggest that enzymes involved in the conversion of glucose to lipid may be targets for the treatment of pathologic cell growth.

The molecular determinants of de novo nucleotide biosynthesis in cancer cells

Tumor cells increase the use of anabolic pathways to satisfy the metabolic requirements associated with a high growth rate. Transformed cells take up and metabolize nutrients such as glucose and glutamine at high levels that support anabolic growth. Oncogenic signaling through the PI3K/Akt and Myc pathways directly control glucose and glutamine uptake, respectively. In order to achieve elevated rates of nucleotide biosynthesis, neoplastic cells must divert carbon from PI3K/Akt-induced glycolytic flux into the nonoxidative branch of the pentose phosphate pathway to generate ribose-5-phosphate. This redirection of glucose catabolism appears to be regulated by cytoplasmic tyrosine kinases. Myc-induced glutamine metabolism also increases the abundance and activity of different rate-limiting enzymes that produce the molecular precursors required for de novo nucleotide synthesis. In this review, we will focus on recent progress in understanding how glucose and glutamine metabolism is redirected by oncogenes in order to support de novo nucleotide biosynthesis during proliferation and how metabolic reprogramming can be potentially exploited in the development of new cancer therapies.

Imatinib resistance associated with BCR-ABL upregulation is dependent on HIF-1alpha-induced metabolic reprograming.

As chronic myeloid leukemia (CML) progresses from the chronic phase to blast crisis, the levels of BCR-ABL increase. In addition, blast-transformed leukemic cells display enhanced resistance to imatinib in the absence of BCR-ABL-resistance mutations. In this study, we show that when BCR-ABL-transformed cell lines were selected for imatinib resistance in vitro, the cells that grew out displayed a higher BCR-ABL expression comparable to the increase seen in accelerated forms of the disease. This enhanced expression of BCR-ABL was associated with an increased rate of glycolysis but with a decreased rate of proliferation. The higher level of BCR-ABL expression in the selected cells correlated with a nonhypoxic induction of hypoxia-inducible factor-1α (HIF-1α) that was required for cells to tolerate enhanced BCR-ABL signaling. HIF-1α induction resulted in an enhanced rate of glycolysis but with reduced glucose flux through both the tricarboxylic acid cycle and the oxidative arm of the pentose phosphate pathway (PPP). The reduction in oxidative PPP-mediated ribose synthesis was compensated by the HIF-1α-dependent activation of the nonoxidative PPP enzyme, transketolase, in imatinib-resistant CML cells. In both primary cultures of cells from patients exhibiting blast transformation and in vivo xenograft tumors, use of oxythiamine, which can inhibit both the pyruvate dehydrogenase complex and transketolase, resulted in enhanced imatinib sensitivity of tumor cells. Together, these results suggest that oxythiamine can enhance imatinib efficacy in patients who present an accelerated form of the disease.

The glucose-responsive transcription factor ChREBP contributes to glucose-dependent anabolic synthesis and cell proliferation

Tumor cells are metabolically reprogrammed to fuel cell proliferation. Most transformed cells take up high levels of glucose and produce ATP through aerobic glycolysis. In cells exhibiting aerobic glycolysis, a significant fraction of glucose carbon is also directed into de novo lipogenesis and nucleotide biosynthesis. The glucose-responsive transcription factor carbohydrate responsive element binding protein (ChREBP) was previously shown to be important for redirecting glucose metabolism in support of lipogenesis in nonproliferating hepatocytes. However, whether it plays a more generalized role in reprogramming metabolism during cell proliferation has not been examined. Here, we demonstrated that the expression of ChREBP can be induced in response to mitogenic stimulation and that the induction of ChREBP is required for efficient cell proliferation. Suppression of ChREBP resulted in diminished aerobic glycolysis, de novo lipogenesis, and nucleotide biosynthesis, but stimulated mitochondrial respiration, suggesting a metabolic switch from aerobic glycolysis to oxidative phosphorylation. Cells in which ChREBP was suppressed by RNAi exhibited p53 activation and cell cycle arrest. In vivo, suppression of ChREBP led to a p53-dependent reduction in tumor growth. These results demonstrate that ChREBP plays a key role both in redirecting glucose metabolism to anabolic pathways and suppressing p53 activity.

Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and overcome drug resistance. A deeper understanding of how a given chemotherapy affects tumor cell death is needed to develop strategically designed anticancer agents. Here, we use a xenograft mouse tumor system generated from genetically defined cells deficient in apoptosis to examine the involvement of multiple forms of cell death induced by cyclophosphamide (CP), a DNA alkylating agent commonly used in chemotherapy. We find that although apoptosis facilitates tumor regression, it is dispensable for complete tumor regression as other forms of cell death are activated. Sporadic necrosis is observed in both apoptosis-competent and deficient tumors evident by tumor cell morphology, extracellular release of high mobility group box 1 protein, and activation of innate immune cells in CP-treated tumors. Our findings indicate that in apoptosis-deficient tumors, necrosis may play a fundamental role in tumor clearance by stimulating the innate immune response.

Abstract 2513: Molecular dissection of cell death mechanisms following ionizing radiation in vitro and in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: A majority of human cancers have the ability to evade cell death with dysregulated cell death and survival mechanisms. This may have a direct impact on treatment resistance and tumor recurrence. This study aims to elucidate the roles of apoptosis, autophagy and necrosis induced by ionizing radiation (IR). Materials/Methods: Immortalized murine embryonic fibroblast (MEF) cell lines with various genetic backgrounds were used in the study. Xenograft tumors were generated by injecting tumor cells (engineered by retrovirally transfecting the above MEF cell lines with oncoprotein E1A and KRAS) to bilateral flanks of nude mice. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after treatments were analyzed by phase contrast microscopy as well as transmission electron microscopy (TEM). Western blotting and Immunohistochemistry (IHC) methods were carried out as described. Results: There was no significant difference in the level of tumor regression between xenografted tumors lacking Bax and Bak (Bax−/−Bak−/−) and WT tumors after radiation. While apoptotic cell death was observed in immortalized WT MEF cell line and xenograft tumors after irradiation, the intrinsic apoptosis pathway was not required Autophagy was found to be activated after radiation, and pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival fraction after radiation, suggesting a cytotoxic role of autophagy following radiation. However, there was no difference in radiation response when an essential autophagy gene ATG5 was stably knocked down, suggesting that autophagy activation may not be required for radiation induced cell death. We then investigated the role of poly(ADP)-ribose polymerase (PARP) in radiation response by using PARP-1−/− immortalized MEFs. These cells were more resistant to MAF (an active metabolite of cyclophosphamide) induced necrosis as was shown previously, but were more sensitive to radiation both in vitro and in vivo (as in xenograft). We also found that a specific PARP inhibitor ABT-888 can significantly sensitize xenograft tumors to radiation treatment. Conclusion: While apoptosis and autophagy occur after radiation, the intrinsic apoptosis and autophagic pathways is not required for tumor regression following IR. Conversely, PARP protein plays an important pro-survival role after IR, and deficiency of PARP renders cells more sensitive to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2513. doi:10.1158/1538-7445.AM2011-2513