Yunhua Zhao

The Effects of Varying Dietary Carbohydrate and Fat Content on Survival in a Murine LNCaP Prostate Cancer Xenograft Model

Purpose: Numerous dietary factors elevate serum levels of insulin and insulin-like growth factor I (IGF-I), both potent prostate cancer mitogens. We tested whether varying dietary carbohydrate and fat, without energy restriction relative to comparison diets, would slow tumor growth and reduce serum insulin, IGF-I, and other molecular mediators of prostate cancer in a xenograft model. Experimental Design: Individually caged male severe combined immunodeficient mice (n = 130) were randomly assigned to one of three diets (described as percent total calories): very high-fat/no-carbohydrate ketogenic diet (NCKD: 83% fat, 0% carbohydrate, 17% protein), low-fat/high-carbohydrate diet (LFD: 12% fat, 71% carbohydrate, 17% protein), or high-fat/moderate-carbohydrate diet (MCD: 40% fat, 43% carbohydrate, 17% protein). Mice were fed to maintain similar average body weights among groups. Following a preliminary feeding period, mice were injected with 1 × 106 LNCaP cells (day 0) and sacrificed when tumors were ≥1,000 mm3. Results: Two days before tumor injection, median NCKD body weight was 2.4 g (10%) and 2.1 g (8%) greater than the LFD and MCD groups, respectively (P < 0.0001). Diet was significantly associated with overall survival (log-rank P = 0.004). Relative to MCD, survival was significantly prolonged for the LFD (hazard ratio, 0.49; 95% confidence interval, 0.29-0.79; P = 0.004) and NCKD groups (hazard ratio, 0.59; 95% confidence interval, 0.37-0.93; P = 0.02). Median serum insulin, IGF-I, IGF-I/IGF binding protein-1 ratio, and IGF-I/IGF binding protein-3 ratio were significantly reduced in NCKD relative to MCD mice. Phospho-AKT/total AKT ratio and pathways associated with antiapoptosis, inflammation, insulin resistance, and obesity were also significantly reduced in NCKD relative to MCD tumors. Conclusions: These results support further preclinical exploration of carbohydrate restriction in prostate cancer and possibly warrant pilot or feasibility testing in humans.

Metformin abolishes increased tumor 18F-2-fluoro-2-deoxy-D-glucose uptake associated with a high energy diet

Insulin regulates glucose uptake by normal tissues. Although there is evidence that certain cancers are growth-stimulated by insulin, the possibility that insulin influences tumor glucose uptake as assessed by 18F-2-Fluoro-2-Deoxy-d-Glucose Positron Emission Tomography (FDG-PET) has not been studied in detail. We present a model of diet-induced hyperinsulinemia associated with increased insulin receptor activation in neoplastic tissue and with increased tumor FDG-PET image intensity. Metformin abolished the diet-induced increases in serum insulin level, tumor insulin receptor activation and tumor FDG uptake associated with the high energy diet but had no effect on these measurements in mice on a control diet. These findings provide the first functional imaging correlate of the well-known adverse effect of caloric excess on cancer outcome. They demonstrate that, for a subset of neoplasms, diet and insulin are variables that affect tumor FDG uptake and have implications for design of clinical trials of metformin as an antineoplastic agent.

Serine Deprivation Enhances Antineoplastic Activity of Biguanides

Metformin, a biguanide widely used in the treatment of type II diabetes, clearly exhibits antineoplastic activity in experimental models and has been reported to reduce cancer incidence in diabetics. There are ongoing clinical trials to evaluate its antitumor properties, which may relate to its fundamental activity as an inhibitor of oxidative phosphorylation. Here, we show that serine withdrawal increases the antineoplastic effects of phenformin (a potent biguanide structurally related to metformin). Serine synthesis was not inhibited by biguanides. Instead, metabolic studies indicated a requirement for serine to allow cells to compensate for biguanide-induced decrease in oxidative phosphorylation by upregulating glycolysis. Furthermore, serine deprivation modified the impact of metformin on the relative abundance of metabolites within the citric acid cycle. In mice, a serine-deficient diet reduced serine levels in tumors and significantly enhanced the tumor growth-inhibitory actions of biguanide treatment. Our results define a dietary manipulation that can enhance the efficacy of biguanides as antineoplastic agents that target cancer cell energy metabolism.

IGF1/insulin receptor kinase inhibition by BMS-536924 is better tolerated than alloxan-induced hypoinsulinemia and more effective than metformin in the treatment of experimental insulin-responsive breast cancer

Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin responsive, but it is unclear whether safe and effective therapies that target the insulin receptor (IR), which is homologous to oncogenes of the tyrosine kinase class, can be developed. We demonstrate that both pharmacologic inhibition of IR family tyrosine kinase activity and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer. Unexpectedly, in contrast to insulin deficiency, pharmacologic IR family inhibition does not lead to significant hyperglycemia and is well tolerated. We show that pharmacokinetic factors explain the tolerability of receptor inhibition relative to insulin deficiency, as the small molecule receptor kinase inhibitor BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers insulin levels only in settings of hyperinsulinemia, had minimal activity in this normoinsulinemic model. These findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors and suggest that therapeutic targeting of the IR family for cancer treatment is practical.

Abstract 4615: The hyperinsulinemia caused by PI3K inhibitors attenuates their antineoplastic efficacy, but can be minimized by co-administration of metformin.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC As phosphatidylinositol 3-kinase (PI3K) signaling is often inappropriately activated in cancer, targeting this pathway is the goal of many drug development programs. PI3K activation provides an important proliferative and antiapoptotic signal for many normal and transformed cell types, but in tissues such as liver, muscle and fat that are involved in regulation of blood glucose, PI3K activation is a consequence of insulin receptor activation, and leads to cellular glucose uptake and reduction in circulating glucose concentration. Therefore, effective pharmacologic PI3K blockade perturbs glucose homeostasis, leading to insulin resistance associated with hyperglycemia and/or compensatory hyperinsulinemia. In some patients, PI3K blockade leads to hyperinsulinemia with normoglycemia, suggesting that elevation of insulin level overcomes pharmacologic PI3K inhibition in insulin-responsive tissues to an extent sufficient to normalize blood glucose. To investigate the possibility that increased insulin stimulation can also attenuate the antiproliferative effect of PI3K inhibitors in cancer cells, we first developed an in vivo model which demonstrated that conventional antineoplastic doses of GDC-0941 (a potent and selective inhibitor of class 1 PI3K) leads to insulin resistance and compensatory hyperinsulinemia (20 min post 2 g/kg glucose IP, serum glucose rises from 5.1 ± 0.4 to 15 ± 1.8 mM in control conditions, but from 12.4 ± 1.2 to 29.2 ± 1.8 mM with GDC-0941 treatment). Next, we showed that the activity of GDC-0941 on a variety neoplastic cells in vitro, in terms of suppression of both proliferation and signaling downstream of PI3K, is attenuated when insulin concentration is raised (75 nM GDC-0941 reduces proliferation by 50%, 36%, 29% when insulin is 2, 6, or 12 nM, respectively). Metformin, which is often administered clinically to patients on PI3K inhibitors to reduce hyperglycemia, abolished GDC-0941-induced hyperinsulinemia. Thus, PI3K inhibitor-induced hyperinsulinemia (a) represents pharmacodynamic evidence of target inhibition (at least in tissues concerned with glycemia regulation), (b) attenuates antineoplastic efficacy, particularly when inhibitor concentrations are suboptimal and (c) may be minimized by co-administration of metformin. These findings are relevant to ongoing clinical trials of PI3K inhibitors, and indicate that the magnitude of the input signal to PI3K influences the degree of blockade achieved by PI3K inhibitors. Citation Format: Marie-Jose Blouin, Elena Birman, Yunhua Zhao, Mahvash Zakikhani, Michael N. Pollak. The hyperinsulinemia caused by PI3K inhibitors attenuates their antineoplastic efficacy, but can be minimized by co-administration of metformin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4615. doi:10.1158/1538-7445.AM2013-4615

A NO CARBOHYDRATE DIET SIGNIFICANTLY PROLONGS SURVIVAL IN A PROSTATE CANCER XENOGRAFT MODEL VIA IGF-1 AND GLOBAL GENE EXPRESSION CHANGES

. RESULTS: At two days prior to tumor injection, median NCKD body weight was 2.4g (10%) and 2.1g (8%) greater than the LFD and WD groups, respectively (p<0.0001). Diet was significantly associated with overall survival (log-rank:p=0.004). Relative to WD, survival was significantly prolonged for LFD (hazard ratio=0.49, 95% CI 0.29-0.79, p=0.004) and NCKD (hazard ratio=0.59, 95% CI 0.37-0.93, p=0.02). Serum insulin, IGF-1, IGF-1:IGFBP-1 ratio, and IGF-1:IGFBP-3 ratio were significantly reduced in the NCKD group. The phospho-AKT:total-AKT ratio and pathways associated with anti-apoptosis, inflammation, insulin- resistance, and obesity were also significantly reduced in NCKD tumors. CONCLUSIONS: In this mouse xenograft model, despite heavier body weights, a carbohydrate restricted diet significantly prolonged survival. These results support further exploration of carbohydrate restriction in future preclinical trials and warrant the possibility of pilot/ feasibility testing in humans.

Abstract 2300: Insulin receptor kinase inhibition is better tolerated than hypoinsulinemia and more effective than metformin in treating breast cancer

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Epidemiologic and experimental evidence suggest that a subset of breast cancer is insulin-responsive, but it is unclear if safe and effective therapies that target the insulin receptor can be developed. We demonstrate that both insulin receptor family tyrosine kinase inhibition and insulin deficiency have anti-neoplastic activity in a model of insulin-responsive breast cancer in mice metabolically normal at baseline. In contrast to insulin deficiency, insulin receptor inhibition does not lead to hyperglycemia and is well-tolerated. We show that pharmacokinetic factors explain the safety of receptor inhibition relative to ligand deficiency, as BMS-536924 does not accumulate in muscle at levels sufficient to block insulin-stimulated glucose uptake. Metformin, which lowers the elevated insulin levels present in settings of insulin resistance, had minimal activity in this model. The findings highlight the importance of tissue-specific drug accumulation as a determinant of efficacy and toxicity of tyrosine kinase inhibitors, and suggest that therapeutic targeting of the insulin receptor family for cancer treatment is practical. 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 2300. doi:10.1158/1538-7445.AM2011-2300