Cell non-autonomous regulation of hepatic IGF-1 and neonatal growth by Kinase Suppressor of Ras 2 (KSR2)

Individuals with poor postnatal growth are at risk for cardiovascular and metabolic problems as adults. Here we show that disruption of the molecular scaffold Kinase Suppressor of Ras 2 (KSR2) causes selective inhibition of hepatic GH signaling in neonatal mice with impaired expression of IGF-1 and IGFBP3. ksr2−/− mice are normal size at birth but show a marked increase in FGF21 accompanied by reduced body mass, shortened body length, and reduced bone mineral density (BMD) and content (BMC) first evident during postnatal development. However, disrupting FGF21 in ksr2−/− mice does not normalize mass, length, or bone density and content in fgf21−/−ksr2−/− mice. Body length, BMC and BMD, but not body mass, are rescued by infection of two-day-old ksr2−/− mice with a recombinant adenovirus encoding human IGF-1. Relative to wild-type mice, GH injections reveal a significant reduction in JAK2 and STAT5 phosphorylation in liver, but not in skeletal muscle, of ksr2−/− mice. However, primary hepatocytes isolated from ksr2−/− mice show no reduction in GH-stimulated STAT5 phosphorylation. These data indicate that KSR2 functions in a cell non-autonomous fashion to regulate GH-stimulated IGF-1 expression in the liver of neonatal mice, which plays a key role in the development of body length

Neutrophils are Mediators of Metastatic Prostate Cancer Progression in Bone

Bone metastatic prostate cancer (BM-PCa) significantly reduces overall patient survival and is currently incurable. Current standard immunotherapy showed promising results for PCa patients with metastatic, but less advanced, disease (i.e., fewer than 20 bone lesions) suggesting that PCa growth in bone contributes to response to immunotherapy. We found that: (1) PCa stimulates recruitment of neutrophils, the most abundant immune cell in bone, and (2) that neutrophils heavily infiltrate regions of prostate tumor in bone of BM-PCa patients. Based on these findings, we examined the impact of direct neutrophil-prostate cancer interactions on prostate cancer growth. Bone marrow neutrophils directly induced apoptosis of PCa in vitro and in vivo, such that neutrophil depletion in bone metastasis models enhanced BM-PCa growth. Neutrophil-mediated PCa killing was found to be mediated by suppression of STAT5, a transcription factor shown to promote PCa progression. However, as the tumor progressed in bone over time, neutrophils from late-stage bone tumors failed to elicit cytotoxic effector responses to PCa. These findings are the first to demonstrate that bone-resident neutrophils inhibit PCa and that BM-PCa are able to progress via evasion of neutrophil-mediated killing. Enhancing neutrophil cytotoxicity in bone may present a novel therapeutic option for bone metastatic prostate cancer

Androgen Receptor Inhibition Suppresses Anti-Tumor Neutrophil Response Against Bone Metastatic Prostate Cancer via Regulation of TβRI Expression

Bone metastatic disease of prostate cancer (PCa) is incurable and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone neutrophils initially inhibit bone metastatic PCa growth yet metastatic PCa becomes resistant to neutrophil response. Further, neutrophils isolated from tumor-bone lost their ability to suppress tumor growth through unknown mechanisms. With this study, our goal was to define the impact of metastatic PCa on neutrophil function throughout tumor progression and to determine the potential of neutrophils as predictive biomarkers of metastatic disease. Using patient peripheral blood polymorphonuclear neutrophils (PMNs), we identified that PCa progression dictates PMN cell surface markers and gene expression, but not cytotoxicity against PCa. Importantly, we also identified a novel phenomenon in which second generation androgen deprivation therapy (ADT) suppresses PMN cytotoxicity via increased transforming growth factor beta receptor I (TβRI). High dose testosterone and genetic or pharmacologic TβRI inhibition rescued androgen receptor-mediated neutrophil suppression and restored neutrophil anti-tumor immune response. These studies highlight the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa

Regulation of Glucose Homeostasis by KSR1 and MARK2

Protein scaffolds control the intensity and duration of signaling and dictate the specificity of signaling through MAP kinase pathways. KSR1 is a molecular scaffold of the Raf/MEK/ERK MAP kinase cascade that regulates the intensity and duration of ERK activation. Relative to wild-type mice, ksr1-/- mice are modestly glucose intolerant, but show a normal response to exogenous insulin. However, ksr1-/- mice also demonstrate a three-fold increase in serum insulin levels in response to a glucose challenge, suggesting a role for KSR1 in insulin secretion. The kinase MARK2 is closely related to C-TAK1, a known regulator of KSR1. Mice lacking MARK2 have an increased rate of glucose disposal in response to exogenous insulin, increased glucose tolerance, and are resistant to diet-induced obesity. mark2-/-ksr1-/- (DKO) mice were compared to wild type, mark2-/-, and ksr1-/- mice for their ability to regulate glucose homeostasis. Here we show that disruption of KSR1 in mark2-/- mice reverses the increased sensitivity to exogenous insulin resulting from MARK2 deletion. DKO mice respond to exogenous insulin similarly to wild type and ksr1-/- mice. These data suggest a model whereby MARK2 negatively regulates insulin sensitivity in peripheral tissue through inhibition of KSR1. Consistent with this model, we found that MARK2 binds and phosphorylates KSR1 on Ser392. Phosphorylation of Ser392 is a critical regulator of KSR1 stability, subcellular location, and ERK activation. These data reveal an unexpected role for the molecular scaffold KSR1 in insulin-regulated glucose metabolism

Coordinating ERK signaling via the molecular scaffold Kinase Suppressor of Ras [version 1; referees: 2 approved]

Many cancers, including those of the colon, lung, and pancreas, depend upon the signaling pathways induced by mutated and constitutively active Ras. The molecular scaffolds Kinase Suppressor of Ras 1 and 2 (KSR1 and KSR2) play potent roles in promoting Ras-mediated signaling through the Raf/MEK/ERK kinase cascade. Here we summarize the canonical role of KSR in cells, including its central role as a scaffold protein for the Raf/MEK/ERK kinase cascade, its regulation of various cellular pathways mediated through different binding partners, and the phenotypic consequences of KSR1 or KSR2 genetic inactivation. Mammalian KSR proteins have a demonstrated role in cellular and organismal energy balance with implications for cancer and obesity. Targeting KSR1 in cancer using small molecule inhibitors has potential for therapy with reduced toxicity to the patient. RNAi and small molecule screens using KSR1 as a reference standard have the potential to expose and target vulnerabilities in cancer. Interestingly, although KSR1 and KSR2 are similar in structure, KSR2 has a distinct physiological role in regulating energy balance. Although KSR proteins have been studied for two decades, additional analysis is required to elucidate both the regulation of these molecular scaffolds and their potent effect on the spatial and temporal control of ERK activation in health and disease

Kinase Suppressor of Ras 2 (KSR2) expression in the brain regulates energy balance and glucose homeostasis

Objective: Kinase Suppressor of Ras 2 (KSR2) is a molecular scaffold coordinating Raf/MEK/ERK signaling that is expressed at high levels in the brain. KSR2 disruption in humans and mice causes obesity and insulin resistance. Understanding the anatomical location and mechanism of KSR2 function should lead to a better understanding of physiological regulation over energy balance. Methods: Mice bearing floxed alleles of KSR2 (KSR2fl/fl) were crossed with mice expressing the Cre recombinase expressed by the Nestin promoter (Nes-Cre) to produce Nes-CreKSR2fl/fl mice. Growth, body composition, food consumption, cold tolerance, insulin and free fatty acid levels, glucose, and AICAR tolerance were measured in gender and age matched KSR2-/- mice. Results: Nes-CreKSR2fl/fl mice lack detectable levels of KSR2 in the brain. The growth and onset of obesity of Nes-CreKSR2fl/fl mice parallel those observed in KSR2-/- mice. As in KSR2-/- mice, Nes-CreKSR2fl/fl are glucose intolerant with elevated fasting and cold intolerance. Male Nes-CreKSR2fl/fl mice are hyperphagic, but female Nes-CreKSR2fl/fl mice are not. Unlike KSR2-/- mice, Nes-CreKSR2fl/fl mice respond normally to leptin and AICAR, which may explain why the degree of obesity of adult Nes-CreKSR2fl/fl mice is not as severe as that observed in KSR2-/- animals. Conclusions: These observations suggest that, in the brain, KSR2 regulates energy balance via control of feeding behavior and adaptive thermogenesis, while a second KSR2-dependent mechanism, functioning through one or more other tissues, modulates sensitivity to leptin and activators of the energy sensor AMPK

MARK2 interacts with KSR1.

<p>A. KSR1-FLAG, C-TAK1-HA, MARK2-HA and their respective vectors were transfected in combination in 293T cells. Thirty-six hours after transfection, cells were lysed and immunoprecipitated with FLAG- and HA-specific antibodies. Proteins were detected on a western blot using antibodies to each epitope tag. B. Schematic of KSR1 constructs used. C. KSR1-FLAG WT or mutants were co-transfected with C-TAK1-HA, MARK2-HA, or empty vectors and cells were lysed and immunoprecipitations performed as in A. IP: immunoprecipitation, WCL: Whole Cell Lysate. * non-specific band.</p

MARK2 phosphorylates KSR1 at Ser392 <i>in vitro</i> and <i>in vivo</i>.

<p>A. KSR1-FLAG, C-TAK1-HA, and MARK2-HA were individually transfected into 293T cells. Thirty-six hours later cells were lysed and HA- or FLAG-immunoprecipitations were performed. KSR1-FLAG immunoprecipitates were phosphatase treated, then incubated with MARK2-HA or C-TAK1-HA immunoprecipitates in the presence of ATP. Western blots were performed and immunoblotted with an anti-pS392 KSR1 specific antibody, anti-KSR1 antibody, or anti-HA antibody. B. Quantification of pS392 KSR1/total KSR1 from panel A, normalized to phosphatase treated WT KSR1 control. Results are the mean +/- S.D. of three independent experiments. C. KSR1-FLAG, C-TAK1-HA, MARK2-HA and their respective empty vectors were transfected in combination in 293T cells. Thirty-six hours after transfection cells were lysed. Proteins were detected on a western blot using antibodies to pS392KSR1, KSR1, or epitope tag of MARK2 and C-TAK1. *p<0.05 compared to ppase treated control.</p

Deletion of KSR1 in <i>mark2^-/-</i> mice reverts insulin sensitivity, but not glucose tolerance.

<p>A. Insulin tolerance tests (ITT) were performed on WT, <i>mark2^-/</i>^-, <i>ksr1^-/-</i> and DKO mice. Results shown are normalized to initial blood glucose levels. B. Glucose tolerance tests (GTT) were performed on WT, <i>mark2^-/</i>^-, <i>ksr1^-/-</i> and DKO mice. Results shown are normalized to initial blood glucose levels. C. Serum insulin levels before GTT (0 min) and 15 min after an IP injection of glucose (15 min). The number of mice analyzed under each condition is indicated above each bar. D. Insulin stimulated ERK activation in BAT. Mice were injected with insulin then sacrificed 15 min later. BAT was excised, lysed, and western blot performed using pERK and ERK specific antibodies. *p<0.05, ***p<0.001.</p

Deletion of KSR1 in <i>mark2^-/-</i> mice does not revert their growth defect.

<p>A. Body weights of WT (black circle), <i>mark2^-/-</i> (grey square), <i>ksr1^-/-</i> (black triangle) and DKO mice (grey triangle) from weaning until 12 weeks of age. B. Adiposity of 12–16 week old mice, determined by DEXA. C. Hematoxylin and eosin stain of WAT and BAT from WT, <i>mark2</i>^-/-, <i>ksr1^-/-</i> and DKO mice. *p<0.05 **p<0.01 ***p<0.001 compared to WT controls.</p