FAK Expression, Not Kinase Activity, Is a Key Mediator of Thyroid Tumorigenesis and Protumorigenic Processes.

UNLABELLED: There are limited therapy options for advanced thyroid cancer, including papillary and anaplastic thyroid cancer (PTC and ATC). Focal adhesion kinase (FAK) regulates cell signaling by functioning as a scaffold and kinase. Previously, we demonstrated that FAK is overexpressed and activated in thyroid cancer cells and human PTC clinical specimens. However, it remains unclear whether patients with advanced thyroid cancer will benefit from FAK inhibition. Therefore, the dual functions of FAK in mediating protumorigenic processes and thyroid tumorigenesis were investigated. Evidence here shows that FAK expression predominantly regulates thyroid cancer cell growth, viability, and anchorage-independent growth. FAK inhibition, with PF-562,271 treatment, modestly reduced tumor volumes, while FAK depletion, through shRNA knockdown, significantly reduced tumor volumes in vivo A role for FAK expression in tumor establishment was demonstrated in a model of PTC, where FAK knockdown tumors did not develop. FAK depletion also led to a significant decrease in overall metastatic burden. Interestingly, pretreatment with a FAK inhibitor resulted in a paradoxical increase in metastasis in a model of ATC, but decreased metastasis in a model of PTC. These data provide the first evidence that FAK expression is critical for the regulation of thyroid tumorigenic functions. IMPLICATIONS: This study demonstrates that FAK expression, but not kinase activity alone, predominantly mediates thyroid tumor growth and metastasis, indicating that targeting the scaffolding function(s) of FAK may be an important therapeutic strategy for advanced thyroid cancer, as well as other FAK-dependent tumors. Mol Cancer Res; 14(9); 869-82. ©2016 AACR.

Skeletal muscle phosphatidylcholine and phosphatidylethanolamine are related to insulin sensitivity and respond to acute exercise in humans.

Several recent reports indicate that the balance of skeletal muscle phosphatidylcholine (PC) and phosphatidylethanolamine (PE) is a key determinant of muscle contractile function and metabolism. The purpose of this study was to determine relationships between skeletal muscle PC, PE and insulin sensitivity, and whether PC and PE are dynamically regulated in response to acute exercise in humans. Insulin sensitivity was measured via intravenous glucose tolerance in sedentary obese adults (OB; n = 14), individuals with type 2 diabetes (T2D; n = 15), and endurance-trained athletes (ATH; n = 15). Vastus lateralis muscle biopsies were obtained at rest, immediately after 90 min of cycle ergometry at 50% maximal oxygen consumption (V̇o2 max), and 2-h postexercise (recovery). Skeletal muscle PC and PE were measured via infusion-based mass spectrometry/mass spectrometry analysis. ATH had greater levels of muscle PC and PE compared with OB and T2D (P < 0.05), with total PC and PE positively relating to insulin sensitivity (both P < 0.05). Skeletal muscle PC:PE ratio was elevated in T2D compared with OB and ATH (P < 0.05), tended to be elevated in OB vs. ATH (P = 0.07), and was inversely related to insulin sensitivity among the entire cohort (r = -0.43, P = 0.01). Muscle PC and PE were altered by exercise, particularly after 2 h of recovery, in a highly group-specific manner. However, muscle PC:PE ratio remained unchanged in all groups. In summary, total muscle PC and PE are positively related to insulin sensitivity while PC:PE ratio is inversely related to insulin sensitivity in humans. A single session of exercise significantly alters skeletal muscle PC and PE levels, but not PC:PE ratio.

Hepatic glucose sensing is impaired, but can be normalized, in people with impaired fasting glucose.

OBJECTIVE: Abnormal endogenous glucose production (EGP) is a characteristic feature in people with impaired fasting glucose (IFG). We sought to determine whether impaired hepatic glucose sensing contributes to abnormal EGP in IFG and whether it could be experimentally restored. METHODS: Glucose production (rate of appearance; Ra) and flux (glucose cycling) were assessed during a hyperglycemic-euinsulinemic somatostatin clamp with an infusion of [6,6-(2)H2-]glucose and [2-(2)H]glucose before and after enhanced hepatic glucokinase activity via an infusion of low-dose fructose in people with IFG and normal glucose tolerance (NGT). RESULTS: During euglycemia, neither endogenous glucose production [(6,6-(2)H2)-glucose Ra; P = 0.53] or total glucose output (TGO; [2-(2)H]-glucose Ra; P = .12) was different between groups, but glucose cycling ([2-(2)H]glucose Ra to [6,6-(2)H2-]glucose Ra; a surrogate measure of hepatic glucokinase activity in the postabsorptive state) was lower in IFG than NGT (P = .04). Hyperglycemia suppressed EGP more in NGT than IFG (P < .01 for absolute or relative suppression, NGT vs IFG), whereas TGO decreased similarly in both groups (P = .77). The addition of fructose completely suppressed EGP in IFG (P < .01) and tended to do the same to TGO (P = .01; no such changes in NGT, P = .39-.55). Glucose cycling (which reflects glucose-6-phosphatase activity during glucose infusion) was similar in IFG and NGT (P = .51) during hyperglycemia and was unchanged and comparable between groups with the addition of fructose (P = .24). CONCLUSIONS: In summary, glucose sensing is impaired in IFG but can be experimentally restored with low-dose fructose. Glucokinase activation may prove to be a novel strategy for the prevention of diabetes in this high-risk group.

Bisphenol A impairs hepatic glucose sensing in C57BL/6 male mice.

AIMS/HYPOTHESIS: Glucose sensing (eg. glucokinase activity) becomes impaired in the development of type 2 diabetes, the etiology of which is unclear. Estrogen can stimulate glucokinase activity, whereas the pervasive environmental pollutant bisphenol A (BPA) can inhibit estrogen action, hence we aimed to determine the effect of BPA on glucokinase activity directly. METHODS: To evaluate a potential acute effect on hepatic glucokinase activity, BPA in water (n = 5) vs. water alone (n = 5) was administered at the EPA's purported "safe dose" (50 µg/kg) by gavage to lean 6-month old male C57BL/6 mice. Two hours later, animals were euthanized and hepatic glucokinase activity measured over glucose levels from 1-20 mmol/l in liver homogenate. To determine the effect of chronic BPA exposure on hepatic glucokinase activity, lean 6-month old male C57BL/6 mice were provided with water (n = 15) or water with 1.75 mM BPA (∼50 µg/kg/day; n = 14) for 2 weeks. Following the 2-week exposure, animals were euthanized and glucokinase activity measured as above. RESULTS: Hepatic glucokinase activity was signficantly suppressed after 2 hours in animals given an oral BPA bolus compared to those who received only water (p = 0.002-0.029 at glucose 5-20 mmol/l; overall treatment effect p<0.001). Exposure to BPA over 2 weeks also suppressed hepatic glucokinase activity in exposed vs. unexposed mice (overall treatment effect, p = 0.003). In both experiments, the Hill coefficient was higher and Vmax lower in mice treated with BPA. CONCLUSIONS/INTERPRETATION: Both acute and chronic exposure to BPA significantly impair hepatic glucokinase activity and function. These findings identify a potential mechanism for how BPA may increase risk for diabetes.

Distinct genetic alterations in the mitogen-activated protein kinase pathway dictate sensitivity of thyroid cancer cells to mitogen-activated protein kinase kinase 1/2 inhibition.

BACKGROUND: The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway plays an important role in papillary and anaplastic thyroid cancer (PTC and ATC) due to activating mutations in BRAF, RAS, or rearrangements in RET/PTC1. The objective of this study was to thoroughly test whether the BRAF V600E mutation predicts response to mitogen-activated protein kinase kinase 1/2 (MKK1/2) inhibition, as shown in other tumor types, using an authenticated panel of thyroid cancer cell lines. METHODS: PTC and ATC cells harboring distinct mutations in the MAPK pathway were treated with two different inhibitors selective for MKK1/2 (CI-1040 or U0126). The consequences of MKK1/2 inhibition on cell growth, survival, invasion, and MAPK signaling was determined. RESULTS: Inhibition of MKK1/2 using CI-1040 or U0126 differentially inhibits the growth of a panel of PTC and ATC cell lines in two-dimensional culture, with those harboring the BRAF V600E mutation (SW1736) or BRAF-V600E/PI3K-E542K mutations (K1) being the most sensitive, the RET/PTC1 rearrangement (TPC1) and BRAF V600E mutant (BCPAP), intermediate, and the HRAS-G13R mutant (C643), the least sensitive. Growth of these cells is more sensitive to MKK1/2 inhibition when grown in 2% versus 10% serum. Baseline levels of phospho-ERK1/2 were similar in all of the cell lines, and inhibition phospho-ERK1/2 did not predict sensitivity to MKK1/2 inhibition. When cells are grown in three-dimensional culture, MKK1/2 inhibition of growth correlates with mutational status (BRAF > RET/PTC1 > RAS). Finally, PTC and ATC invasiveness is differentially inhibited by CI-1040, which is independent of tumor type or mutation present. CONCLUSIONS: Different mutations in the MAPK pathway play distinct roles in the growth and invasion of thyroid cancer cells. These results indicate that MKK1/2 inhibitors have the potential to inhibit thyroid cancer growth and invasion, but that responses differ based on mutation status and growth conditions.

Inhibition of Src with AZD0530 reveals the Src-Focal Adhesion kinase complex as a novel therapeutic target in papillary and anaplastic thyroid cancer.

CONTEXT: Focal adhesion kinase (FAK) and Src are overexpressed and activated in many cancers and have been associated with tumor progression. The role of the Src-FAK complex has not been characterized in papillary and anaplastic thyroid cancer (PTC and ATC). OBJECTIVE: The goal of this study was to determine the role of Src and FAK in the growth and invasion of PTC and ATC. DESIGN: PTC and ATC cells were treated with the oral Src inhibitor, AZD0530, to determine the consequences of Src inhibition using growth and invasion assays. FAK and phospho-FAK levels were analyzed in cell lines as well as in PTC tumor samples. RESULTS: AZD0530 treatment inhibited the growth and invasion in four of five thyroid cancer cell lines, and inhibition did not correlate with basal levels of phospho-Src. Instead, we show for the first time that FAK, a critical substrate and effector of Src, is phosphorylated at tyrosine residue 861 (pY861) in PTC and ATC cells, and high levels of phospho-FAK correlate with AZD0530 sensitivity. We further showed that pY861-FAK phosphorylation is Src-dependent. Sensitivity to AZD0530 was confirmed using a preclinical three-dimensional culture model. Phospho-ERK1/2 was not affected by AZD0530, indicating that Src signaling does not require MAPK. Finally, FAK and pY861-FAK were expressed in 10 of 10 and five of 10 PTC tumors, respectively. CONCLUSIONS: Inhibition of the Src-FAK complex represents a promising therapeutic strategy for patients with advanced thyroid cancer, and phospho-FAK represents a potential biomarker for response.