The Role of the Insulin/IGF System in Cancer: Lessons Learned from Clinical Trials and the Energy Balance-Cancer Link

Numerous epidemiological and pre-clinical studies have demonstrated that the insulin/insulin-like growth factor (IGF) system plays a key role in the development and progression of several types of cancer. Insulin/IGF signaling, in cooperation with chronic low-grade inflammation, is also an important contributor to the cancer-promoting effects of obesity. However, clinical trials for drugs targeting different components of this system have produced largely disappointing results, possibly due to the lack of predictive biomarker use and problems with the design of combination therapy regimens. With careful attention to the identification of likely patient responders and optimal drug combinations, the outcome of future trials may be improved. Given that insulin/IGF signaling is known to contribute to obesity-associated cancer, further investigation regarding the efficacy of drugs targeting this system and its downstream effectors in the obese patient population is warranted

Obesity Suppresses Estrogen Receptor Beta Expression in Breast Cancer Cells via a HER2-Mediated Pathway

Obesity is associated with a worse breast cancer prognosis, while greater breast tumor estrogen receptor beta (ERβ) expression is correlated with improved therapy response and survival. The objective of this study was to determine the impact of obesity on breast cancer cell ERβ expression, which is currently unknown. We utilized an in vitro model of obesity in which breast cancer cells were exposed to patient serum pooled by body mass index category (obese (OB): ≥30 kg/m2; normal weight (N): 18.5–24.9 kg/m2). Four human mammary tumor cell lines representing the major breast cancer subtypes (SKBR3, MCF-7, ZR75, MDA-MB-231) and mammary tumor cells from MMTV-neu mice were used. ERβ expression, assessed by qPCR and western blotting, was suppressed in the two HER2-overexpressing cell lines (SKBR3, MMTV-neu) following OB versus N sera exposure, but did not vary in the other cell lines. Expression of Bcl-2 and cyclin D1, two genes negatively regulated by ERβ, was elevated in SKBR3 cells following exposure to OB versus N sera, but this difference was eliminated when the ERβ gene was silenced with siRNA. Herceptin, a HER2 antagonist, and siRNA to HER2 were used to evaluate the role of HER2 in sera-induced ERβ modulation. SKBR3 cell treatment with OB sera plus Herceptin increased ERβ expression three-fold. Similar results were obtained when HER2 expression was silenced with siRNA. OB sera also promoted greater SKBR3 cell viability and growth, but this variance was not present when ERβ was silenced or the cells were modified to overexpress ERβ. Based on this data, we conclude that obesity-associated systemic factors suppress ERβ expression in breast cancer cells via a HER2-mediated pathway, leading to greater cell viability and growth. Elucidation of the mechanism(s) mediating this effect could provide important insights into how ERβ expression is regulated as well as how obesity promotes a more aggressive disease

Targeting cholesterol-rich microdomains to circumvent tamoxifen-resistant breast cancer

Adjuvant treatment with tamoxifen substantially improves survival of women with estrogen-receptor positive (ER+) tumors. Tamoxifen resistance (TAMR) limits clinical benefit. RRR alpha tocopherol ether-linked acetic acid analogue (alpha-TEA) is a small bioactive lipid with potent anticancer activity. We evaluated the ability of alpha-TEA in the presence of tamoxifen to circumvent TAMR in human breast cancer cell lines. Methods: Two genotypically matched sets of TAM-sensitive (TAMS) and TAM-resistant (TAMR) human breast cancer cell lines were assessed for signal-transduction events with Western blotting, apoptosis induction with Annexin V-FITC/PI assays, and characterization of cholesterol-rich microdomains with fluorescence staining. Critical involvement of selected mediators was determined by using RNA interference and chemical inhibitors. Results: Growth-factor receptors (total and phosphorylated forms of HER-1 and HER-2), their downstream prosurvival mediators pAkt, pmTOR, and pERK1/2, phosphorylated form of estrogen receptor-alpha (pER-alpha at Ser-167 and Ser-118, and cholesterol-rich lipid microdomains were highly amplified in TAMR cell lines and enhanced by treatment with TAM. alpha-TEA disrupted cholesterol-rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators, and induced DR5-mediated mitochondria-dependent apoptosis via an endoplasmic reticulum stress-triggered pro-death pJNK/CHOP/DR5 amplification loop. Furthermore, methyl-beta-cyclodextrin (M beta CD), a chemical disruptor of cholesterol rich microdomains, acted cooperatively with TAM to reduce prosurvival mediators and to induce apoptosis. Conclusions: Data for the first time document that targeting cholesterol-rich lipid microdomains is a potential strategy to circumvent TAMR, and the combination of alpha-TEA + TAM can circumvent TAMR by suppression of prosurvival signaling via disruption of cholesterol-rich lipid microdomains and activation of apoptotic pathways via induction of endoplasmic reticulum stress.Clayton Foundation for ResearchCenter for Molecular and Cellular Toxicology at the University of TexasNIEHS/NIH T32 ES07247Nutritional Science

Omega-3-Acid Ethyl Esters Block the Protumorigenic Effects of Obesity in Mouse Models of Postmenopausal Basal-like and Claudin-Low Breast Cancer

Obesity induces chronic inflammation and is an established risk and progression factor for triple-negative breast cancers, including basal-like (BL) and claudin-low (CL) subtypes. We tested the effects of dietary supplementation with ethyl esters of the marine-derived anti-inflammatory omega-3 fatty acids eicosapentaenoic and docosahexaenoic acid (EPA+DHA; Lovaza®) on growth of murine BL and CL mammary tumors. Female ovariectomized C57BL/6 mice were fed a control diet or a diet-induced obesity (DIO) diet with or without EPA+DHA (0.025%, resulting in blood levels of EPA and DHA comparable to women taking Lovaza 4 g/day) for 6 weeks. All mice were then orthotopically injected with Wnt-1 cells (a BL tumor cell suspension derived from MMTV-Wnt-1 transgenic mouse mammary tumors) or M-Wnt cells (a CL tumor cell line cloned from the Wnt-1 tumor cell suspension). Mice were killed when tumors were 1 cm in diameter. EPA+DHA supplementation did not significantly impact Wnt-1 or M-Wnt mammary tumor growth in normoweight control mice. However, EPA+DHA supplementation in DIO mice reduced growth of Wnt-1 and M-Wnt tumors; reduced leptin:adiponectin ratio and pro-inflammatory eicosanoids in the serum; improved insulin sensitivity; and decreased tumoral expression of cyclooxygenase-2 and phospho-p65. Thus, EPA+DHA supplementation in mouse models of postmenopausal BL and CL breast cancer offsets many of the pro-tumorigenic effects of obesity. These preclinical findings, in combination with results from parallel biomarker studies in women, suggest EPA+DHA supplementation may reduce the burden of BL and CL breast cancer in obese women

PDK-1/AKT pathway as a novel therapeutic target in rhabdomyosarcoma cells using OSU-03012 compound

Rhabdomyosarcoma (RMS) is the most common paediatric soft-tissue sarcoma including two major subtypes, alveolar rhabdomyosarcoma (ARMS) and embryonal rhabdomyosarcoma (ERMS). Increasing evidence suggests that oncogenesis of RMS involves multistages of signalling protein dysregulation which may include prolonged activation of serine/threonine kinases such as phosphoinositide-dependant kinase-1 (PDK-1) and AKT. To date, whether PDK-1/AKT pathway is activated in RMS is unknown. This study was to examine phosphorylation status of AKT and to evaluate a novel small molecular inhibitor, OSU-03012 targeting PDK-1 in RMS. We examined phosphorylation levels of AKT using ARMS and ERMS tissue microarray and immunohistochemistry staining. Our results showed phospho-AKTThr308 level is elevated 42 and 35% in ARMS and ERMS, respectively. Phospho-AKTSer473 level is also increased 43% in ARMS and 55% in ERMS. Furthermore, we showed that OSU-03012 inhibits cell viability and induces apoptosis in ARMS and ERMS cell lines (RH30, SMS-CTR), which express elevated phospho-AKT levels. Normal cells are much less sensitive to OSU-03012 and in which no detectable apoptosis was observed. This study showed, for the first time, that PDK-1/AKT pathway is activated in RMS and may play an important role in survival of RMS. PDK-1/AKT pathway may be an attractive therapeutic target for cancer intervention in RMS using OSU-03012

New targets for therapy in breast cancer: Mammalian target of rapamycin (mTOR) antagonists

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase member of the cellular phosphatidylinositol 3-kinase (PI3K) pathway, which is involved in multiple biologic functions such as transcriptional and translational control. mTOR is a downstream mediator in the PI3K/Akt signaling pathway and plays a critical role in cell survival. In breast cancer this pathway can be activated by membrane receptors, including the HER (or ErbB) family of growth factor receptors, the insulin-like growth factor receptor, and the estrogen receptor. There is evidence suggesting that Akt promotes breast cancer cell survival and resistance to chemotherapy, trastuzumab, and tamoxifen. Rapamycin is a specific mTOR antagonist that targets this pathway and blocks the downstream signaling elements, resulting in cell cycle arrest in the G(1 )phase. Targeting the Akt/PI3K pathway with mTOR antagonists may increase the therapeutic efficacy of breast cancer therapy

Energy Balance Modulation Impacts Epigenetic Reprogramming, ERα and ERβ Expression, and Mammary Tumor Development in MMTV-neu Transgenic Mice

The association between obesity and breast cancer risk and prognosis is well established in ER-positive disease but less clear in HER2-positive disease. Here, we report preclinical evidence suggesting weight maintenance through calorie restriction may limit risk of HER2-positive breast cancer. In female MMTV-HER2/neu transgenic mice, we found that ERα and ERβ expression, mammary tumorigenesis and survival are energy balance-dependent in association with epigenetic reprogramming. Mice were randomized to receive a calorie restriction (CR), overweight (OW)-inducing, or diet-induced obesity (DIO) regimen (n = 27/group). Subsets of mice (n = 4/group/time point) were euthanized after 1, 3 and 5 months to characterize diet-dependent metabolic, transcriptional, and epigenetic perturbations. Remaining mice were followed up to 22 months. Relative to the OW and DIO regimens, CR decreased body weight, adiposity, and serum metabolic hormones as expected, and also elicited an increase in mammary ERα and ERβ expression. Increased DNA methylation accompanied this pattern, particularly at CpG dinucleotides located within binding or flanking regions for the transcriptional regulator CCCTC-binding factor (CTCF) of ESR1 and ESR2, consistent with sustained transcriptional activation of ERα and ERβ. Mammary expression of the DNA methylation enzyme DNMT1 was stable in CR mice but increased over time in OW and DIO mice, suggesting CR obviates epigenetic alterations concurrent with chronic excess energy intake. In the survival study, CR elicited a significant suppression in spontaneous mammary tumorigenesis. Overall, our findings suggest a mechanistic rationale to prevent or reverse excess body weight as a strategy to reduce HER2-positive breast cancer risk

Inhibitory effect of HGF on invasiveness of aggressive MDA-MB231 breast carcinoma cells, and role of HDACs

Hepatocyte growth factor (HGF), through Met receptor binding, fulfils numerous functions in invasive tumour growth (survival/proliferation, motility, apoptosis), but epigenetic control of gene expression in this process is poorly understood. In HGF-treated breast cancer cells we studied (a) the chemoinvasion towards CXCL12 (ligand of the chemokine-receptor CXCR4) and (b) the mechanistic basis, that is, the transduction pathways that regulate CXCR4-mediated invasion, and the role played by histone deacetylases (HDACs) after blockade with trichostatin A (TSA). In highly invasive and metastatic MDA-MB231 cells HGF had a dual inhibitory effect, reducing spontaneous migration and specific chemoinvasion towards CXCL12, the latter by decreasing CXCR4 transactivation and protein level. After HGF the levels of phosphorylated (therefore active) c-Src and Akt persistently increased, indicating a role of these signal transducers in the HGF-dependent cellular and molecular effects. c-Src wild-type expression vector (Srcwt) increased active c-Src and mimicked the HGF-dependent inhibition of CXCR4 transactivation. Our findings indicate that HDACs participated in the HGF-inhibitory effects. In fact, blockade of HDACs hindered the HGF- and Srcwt-dependent reductions of CXCR4 transactivation and invasiveness, while inhibition of endogenous c-Src was additive with HGF, further reducing specific chemoinvasion. In conclusion, in MDA-MB231 cells HDAC blockade with TSA partly counteracted the HGF-dependent effects through molecular events that included enhancement of the expression of the genes for invasiveness Met and CXCR4 (depending on serum conditions), reduction of endogenous phospho-c-Src/c-Src and phosphoAkt/Akt ratios and triggering of apoptosis. The potential therapeutic use of TSA should take into account the variable aggressiveness of breast carcinoma cells and microenvironment signals such as HGF at the secondary growth site of the tumour. It was interesting that HGF reduced motility and CXCR4 functionality only of MDA-MB231 cells, and not of low-invasive MCF-7 cells, suggesting a mechanism implicated in metastatic cell homing