LaTonia Taliaferro-Smith

Bidirectional Crosstalk between Leptin and Insulin-like Growth Factor-I Signaling Promotes Invasion and Migration of Breast Cancer Cells via Transactivation of Epidermal Growth Factor Receptor

Obesity is an independent risk factor for breast cancer, and obese breast cancer patients exhibit a higher risk for larger tumor burden and increased metastasis. Obesity, as associated with metabolic syndrome, results in an increase in circulating insulin-like growth factor (IGF), which acts as a mitogen. In addition, higher plasma level of adipocytokine leptin is associated with obesity. In the present study, we show that cotreatment with leptin and IGF-I significantly increases proliferation as well as invasion and migration of breast cancer cells. We found a novel bidirectional crosstalk between leptin and IGF-I signaling; IGF-I induced phosphorylation of leptin receptor (Ob-Rb) and leptin induced phosphorylation of IGF-I receptor (IGF-IR), whereas cotreatment induced synergistic phosphorylation and association of Ob-Rb and IGF-IR along with activation of downstream effectors, Akt and extracellular signal-regulated kinase. Leptin increased phosphorylation of IGF signaling molecules insulin-receptor substrate (IRS)-1 and IRS-2. Interestingly, we found that leptin and IGF-I cotreatment synergistically transactivated epidermal growth factor receptor (EGFR), depending on the proteolytic release of EGFR ligands, as the broad-spectrum matrix metalloproteinase inhibitor GM6001 could inhibit this effect. Using clinically relevant EGFR inhibitors, erlotinib and lapatinib, we found that inhibition of EGFR activation effectively inhibited leptin- and IGF-I-induced invasion and migration of breast cancer cells. Taken together, these data suggest a novel bidirectional crosstalk between leptin and IGF-I signaling that transactivates EGFR and promotes the metastatic properties as well as invasion and migration of breast cancer cells. Our findings indicate the possibility of using EGFR inhibitors erlotinib and lapatinib to counter the procancerous effects of leptin and IGF-I in breast cancers.

LKB1 is required for adiponectin-mediated modulation of AMPK-S6K axis and inhibition of migration and invasion of breast cancer cells

Adiponectin is widely known as an adipocytokine with therapeutic potential for its markedly protective function in the pathogenesis of obesity-related disorders, metabolic syndrome, systemic insulin resistance, cardiovascular disease and more recently carcinogenesis. In the present study, we show that adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Further analysis of the underlying molecular mechanisms revealed that adiponectin treatment increased AMP-activated protein kinase (AMPK) phosphorylation and activity as evident by increased phosphorylation of downstream target of AMPK, acetyl-coenzyme A carboxylase and inhibition of p70S6 kinase (S6K). Intriguingly, we discovered that adiponectin treatment increases the expression of tumor suppressor gene LKB1 in breast cancer cells. Overexpression of LKB1 in breast cancer cells further increased adiponectin-mediated phosphorylation of AMPK. Using isogenic LKB1 knockdown cell line pair, we found that LKB1 is required for adiponectin-mediated modulation of AMPK–S6K axis and more importantly, inhibition of adhesion, migration and invasion of breast cancer cells. Taken together these data present a novel mechanism involving specific upregulation of tumor suppressor gene LKB1 by which adiponectin inhibits adhesion, invasion and migration of breast cancer cells. Our findings indicate the possibility of using adiponectin analogues to inhibit invasion and migration of breast cancer cells.

Combinatorial Effects of Lapatinib and Rapamycin in Triple-Negative Breast Cancer Cells

Triple-negative breast cancers, which lack estrogen receptor, progesterone receptor, and HER2/neu overexpression, account for approximately 15% of breast cancers, but occur more commonly in African Americans. The poor survival outcomes seen with triple-negative breast cancers patients are, in part, due to a lack of therapeutic targets. Epidermal growth factor receptor (EGFR) is overexpressed in 50% of triple-negative breast cancers, but EGFR inhibitors have not been effective in patients with metastatic breast cancers. However, mTOR inhibition has been shown to reverse resistance to EGFR inhibitors. We examined the combination effects of mTOR inhibition with EGFR inhibition in triple-negative breast cancer in vitro and in vivo. The combination of EGFR inhibition by using lapatinib and mTOR inhibition with rapamycin resulted in significantly greater cytotoxicity than the single agents alone and these effects were synergistic in vitro. The combination of rapamycin and lapatinib significantly decreased growth of triple-negative breast cancers in vivo compared with either agent alone. EGFR inhibition abrogated the expression of rapamycin-induced activated Akt in triple-negative breast cancer cells in vitro. The combination of EGFR and mTOR inhibition resulted in increased apoptosis in some, but not all, triple-negative cell lines, and these apoptotic effects correlated with a decrease in activated eukaryotic translation initiation factor (eIF4E). These results suggest that mTOR inhibitors could sensitize a subset of triple-negative breast cancers to EGFR inhibitors. Given the paucity of effective targeted agents in triple-negative breast cancers, these results warrant further evaluation. Mol Cancer Ther; 10(8); 1460–9. ©2011 AACR.

Reciprocal regulation of ZEB1 and AR in triple negative breast cancer cells

Zinc-finger enhancer binding protein (ZEB1) is a transcription factor involved in the progression of cancer primarily through promoting epithelial to mesenchymal transition (EMT). ZEB1 represses the expression of E-cadherin by binding to E-box sequences in the promoter, thus decreasing epithelial differentiation. We show that ZEB1 and androgen receptor (AR) cross-talk in triple negative breast cancer cell lines. Chromatin immunoprecipitation analysis demonstrates that ZEB1 binds directly to the E-box located in the AR promoter. ZEB1 suppression by stably transfecting shRNA in a triple negative breast cancer cell line resulted in a decrease of AR mRNA, protein, and AR downstream targets. ZEB1 knockdown in triple negative breast cancer cells sensitized the cells to bicalutamide by reducing migration compared to the control cells. Conversely, blockade of AR signaling with bicalutamide resulted in a suppression of ZEB1 protein expression in two triple negative breast cancer cell lines. Furthermore, using a breast cancer tissue microarray, a majority of triple negative breast cancers exhibit positive staining for both ZEB1 and AR. Taken together, these results indicate that ZEB1 and AR regulate each other to promote cell migration in triple negative breast cancer cells.

Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1α and STAT-3

Hypoxia-inducible factors (HIFs) and STAT-3 play essential roles in angiogenesis. HIF-1α and STAT-3 are clients of the heat shock protein 90 (HSP90). We hypothesized that ganetespib, a potent HSP90 inhibitor, would disrupt angiogenesis in colorectal cancer (CRC) through inhibition of HIF-1α and STAT-3. CRC cell lines (HCT116 and HT29) were used in all the experiments. Egg CAM and HUVEC assays revealed decreased angiogenesis in ganetespib treated cell lines. Ganetespib inhibited matrigel plug vascularization and tumor growth of xenografts. Significant inhibition of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, HIF-1α and STAT-3 expression was observed in both cell lines treated ganetespib. HIF-1α overexpression resulted in the increase VEGF and STAT-3 expression and this was inhibited by ganetespib. HIF-1α knockdown inhibited VEGF and STAT-3 expression. STAT-3 knockdown inhibited VEGF but not HIF-1α expression. HSP90, STAT-3 and VEGF expression was significantly higher in CRC compared to adjacent normal tissue. Significant downregulation of PDGFA, FGF2, Ang-1, Ang-2, TGFβ1, VEGF, STAT-3 and HIF-1α mRNA was observed in the post ganetespib treatment tumor samples from patients with rectal cancer. These results collectively suggest that inhibition of HSP90 is a promising antiangiogenic strategy in CRC. HSP90 angiogenic effects are mediated through HIF-1α and STAT-3.

Snail Promotes Epithelial Mesenchymal Transition in Breast Cancer Cells in Part via Activation of Nuclear ERK2

Snail transcription factor is up-regulated in several cancers and associated with increased tumor migration and invasion via induction of epithelial-to-mesenchymal transition (EMT). MAPK (ERK1/2) signaling regulates cellular processes including cell motility, adhesion, and invasion. We investigated the regulation of ERK1/2 by Snail in breast cancer cells. ERK1/2 activity (p-ERK) was higher in breast cancer patient tissue as compared to normal tissue. Snail and p-ERK were increased in several breast cancer cell lines as compared to normal mammary epithelial cells. Snail knockdown in MDA-MB-231 and T47-D breast cancer cells decreased or re-localized p-ERK from the nuclear compartment to the cytoplasm. Snail overexpression in MCF-7 breast cancer cells induced EMT, increased cell migration, decreased cell adhesion and also increased tumorigenicity. Snail induced nuclear translocation of p-ERK, and the activation of its subcellular downstream effector, Elk-1. Inhibiting MAPK activity with UO126 or knockdown of ERK2 isoform with siRNA in MCF-7 Snail cells reverted EMT induced by Snail as shown by decreased Snail and vimentin expression, decreased cell migration and increased cell adhesion. Overall, our data suggest that ERK2 isoform activation by Snail in aggressive breast cancer cells leads to EMT associated with increased cell migration and decreased cell adhesion. This regulation is enhanced by positive feedback regulation of Snail by ERK2. Therefore, therapeutic targeting of ERK2 isoform may be beneficial for breast cancer.

Agouti-related protein promoter variant associated with leanness and decreased risk for diabetes in West Africans

Objective:The role of the central melanocortin system in the development of obesity has been extensively studied. Single-nucleotide polymorphisms (SNPs) within several candidate genes have been associated with food intake and obesity-related phenotypes; however, few of these associations have been replicated. SNPs in the agouti-related protein (AGRP) gene coding (Ala67Thr, 199G/A) and promoter (−38C/T) have been reported to be associated with body mass index (BMI), fat mass (FM) and percent body fat, in populations of European and African descent. In this study, we evaluated the association between the functional AGRP −38C/T promoter SNP and weight-related traits, namely BMI, FM and fat-free mass (FFM), as well as diabetes status.Design:An association study of the AGRP −38C/T SNP and indices of obesity and diabetes status.Subjects:A well-characterized population of 538 West Africans from Ghana and Nigeria recruited in the AADM (Africa America Diabetes Mellitus) study (mean age 52 years, 41.3% males, 71% diabetic).Measurements:Genotyping of the AGRP −38C/T SNP, BMI, FM, FFM and fasting plasma glucose.Results:Women carrying two copies of the variant T allele had significantly lower BMI (OR=0.47; 95% CI, 0.25–0.87). Also, men with at least one copy of the variant T allele were over two times less likely to be diabetic than other men (OR=0.44; 95% CI, 0.22–0.89).Conclusion:Our results replicate previous findings and implicate the AGRP −38C/T SNP in the regulation of body weight in West Africans.

Heat Shock Protein 90 Promotes Epithelial to Mesenchymal Transition, Invasion, and Migration in Colorectal Cancer

Epithelial to mesenchymal transition (EMT), invasion, and motility are essential steps in colorectal cancer (CRC) metastasis regulated by HIF‐1α and NF‐κB. Since HSP90 activates HIF‐1α and NF‐κB, we hypothesized that inhibition of HSP90 leads to inhibition of HIF‐1α and NF‐κB resulting in inhibition of EMT, invasion, and motility. Treatment of colorectal cancer cell lines HT‐29 and HCT‐116 with ganetespib at 50 nM for 24 h inhibited EMT (downregulated vimentin and upregulated E‐cadherin), matrigel invasion, and spheroid migration. Ganetespib treatment or HSP90 knockdown downregulated molecular pathways associated with EMT, invasion, and motility. The overexpression of HIF‐1α or NF‐κB resulted in increased EMT, invasion, and motility in both cell lines and these effects were inhibited by ganetespib. Similar effects were observed in animal xenografts treated with ganetespib. Taken together, our data demonstrate for the first time that inhibition of HSP90 downregulates both HIF‐1α and NF‐κB leading to inhibition of EMT, motility, and invasiveness in colorectal cancer.

Insulin-like growth factor-1 receptor signaling increases the invasive potential of human epidermal growth factor receptor 2-overexpressing breast cancer cells via Src-focal adhesion kinase and forkhead box protein M1.

Resistance to the human epidermal growth factor receptor (HER2)–targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1–mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1.

Computational analysis of the structural basis of ligand binding to the crustacean retinoid X receptor.

Homodimerization of the retinoid X receptor (RXR) occurs upon binding of ligands to the receptor, but little is known about structural mechanisms involved in RXR ligand binding. In the present study, binding of known ligands (5-Hydroxytryptamine, dopamine and naloxone) to the Celuca pugilator RXR was modeled computationally using the human RXR-α as a homology template. Docking scores calculated for these ligands showed reasonably good binding interactions to C. pugilator RXR. Furthermore, RXR is the receptor that mediates the different activities of neurotransmitters and opioid against naloxone in crustaceans and possibly other species. These results indicate that 5-hydroxytryptamine and naloxone might have similar functions. These also results suggest a 3-D model of C. pugilator RXR that describes the binding of ligands at a single RXR receptor binding site and offers further insight into the binding of structurally diverse ligands to this receptor. Further, computational studies showed that crustacean RXRs might be closer to vertebrate RXR than to insect RXR. The predicted binding models for C. pugilator RXR may allow for better design of experimental studies, such as site-directed mutagenesis and affinity labeling studies that may yield valuable information concerning structure-activity relationship studies of RXR and its ligands.