Age-Related Increases in IGFBP2 Increase Melanoma Cell Invasion and Lipid Synthesis.

Aged patients with melanoma (>65 years old) have more aggressive disease relative to young patients (<55 years old) for reasons that are not completely understood. Analysis of the young and aged secretome from human dermal fibroblasts identified >5-fold levels of IGF-binding protein 2 (IGFBP2) in the aged fibroblast secretome. IGFBP2 functionally triggers upregulation of the PI3K-dependent fatty acid biosynthesis program in melanoma cells. Melanoma cells co-cultured with aged dermal fibroblasts have higher levels of lipids relative to those co-cultured with young dermal fibroblasts, which can be lowered by silencing IGFBP2 expression in fibroblasts prior to treating with conditioned media. Conversely, ectopically treating melanoma cells with recombinant IGFBP2 in the presence of conditioned media from young fibroblasts or overexpressing IGFBP2 in melanoma cells promoted lipid synthesis and accumulation in melanoma cells. Treatment of young mice with rIGFBP2 increases tumor growth. Neutralizing IGFBP2 in vitro reduces migration and invasion in melanoma cells, and in vivo studies demonstrate that neutralizing IGFBP2 in syngeneic aged mice reduces tumor growth and metastasis. Our results suggest that aged dermal fibroblasts increase melanoma cell aggressiveness through increased secretion of IGFBP2, stressing the importance of considering age when designing studies and treatment. SIGNIFICANCE: The aged microenvironment drives metastasis in melanoma cells. This study reports that IGFBP2 secretion by aged fibroblasts induces lipid accumulation in melanoma cells, driving an increase in tumor invasiveness. Neutralizing IGFBP2 decreases melanoma tumor growth and metastasis.

Age-dependent loss of HAPLN1 erodes vascular integrity via indirect upregulation of endothelial ICAM1 in melanoma.

Melanoma, the most lethal form of skin cancer, often has worse outcomes in older patients. We previously demonstrated that an age-related decrease in the secreted extracellular matrix (ECM) protein HAPLN1 has a role in slowing melanoma progression. Here we show that HAPLN1 in the dermal ECM is sufficient to maintain the integrity of melanoma-associated blood vessels, as indicated by increased collagen and VE-cadherin expression. Specifically, we show that HAPLN1 in the ECM increases hyaluronic acid and decreases endothelial cell expression of ICAM1. ICAM1 phosphorylates and internalizes VE-cadherin, a critical determinant of vascular integrity, resulting in permeable blood vessels. We found that blocking ICAM1 reduces tumor size and metastasis in older mice. These results suggest that HAPLN1 alters endothelial ICAM1expression in an indirect, matrix-dependent manner. Targeting ICAM1 could be a potential treatment strategy for older patients with melanoma, emphasizing the role of aging in tumorigenesis.

Fibroblasts in the Aged Pancreas Drive Pancreatic Cancer Progression.

Pancreatic cancer is more prevalent in older individuals and often carries a poorer prognosis for them. The relationship between the microenvironment and pancreatic cancer is multifactorial, and age-related changes in nonmalignant cells in the tumor microenvironment may play a key role in promoting cancer aggressiveness. Because fibroblasts have profound impacts on pancreatic cancer progression, we investigated whether age-related changes in pancreatic fibroblasts influence cancer growth and metastasis. Proteomics analysis revealed that aged fibroblasts secrete different factors than young fibroblasts, including increased growth/differentiation factor 15 (GDF-15). Treating young mice with GDF-15 enhanced tumor growth, whereas aged GDF-15 knockout mice showed reduced tumor growth. GDF-15 activated AKT, rendering tumors sensitive to AKT inhibition in an aged but not young microenvironment. These data provide evidence for how aging alters pancreatic fibroblasts and promotes tumor progression, providing potential therapeutic targets and avenues for studying pancreatic cancer while accounting for the effects of aging. SIGNIFICANCE: Aged pancreatic fibroblasts secrete GDF-15 and activate AKT signaling to promote pancreatic cancer growth, highlighting the critical role of aging-mediated changes in the pancreatic cancer microenvironment in driving tumor progression. See related commentary by Isaacson et al., p. 1185.

Tyrosine kinases compete for growth hormone receptor binding and regulate receptor mobility and degradation.

Growth hormone (GH) acts via JAK2 and LYN to regulate growth, metabolism, and neural function. However, the relationship between these tyrosine kinases remains enigmatic. Through an interdisciplinary approach combining cell biology, structural biology, computation, and single-particle tracking on live cells, we find overlapping LYN and JAK2 Box1-Box2-binding regions in GH receptor (GHR). Our data implicate direct competition between JAK2 and LYN for GHR binding and imply divergent signaling profiles. We show that GHR exhibits distinct mobility states within the cell membrane and that activation of LYN by GH mediates GHR immobilization, thereby initiating its nanoclustering in the membrane. Importantly, we observe that LYN mediates cytokine receptor degradation, thereby controlling receptor turnover and activity, and this applies to related cytokine receptors. Our study offers insight into the molecular interactions of LYN with GHR and highlights important functions for LYN in regulating GHR nanoclustering, signaling, and degradation, traits broadly relevant to many cytokine receptors.

Fibroblasts in cancer: Unity in heterogeneity.

Tumor cells do not exist in isolation in vivo, and carcinogenesis depends on the surrounding tumor microenvironment (TME), composed of a myriad of cell types and biophysical and biochemical components. Fibroblasts are integral in maintaining tissue homeostasis. However, even before a tumor develops, pro-tumorigenic fibroblasts in close proximity can provide the fertile 'soil' to the cancer 'seed' and are known as cancer-associated fibroblasts (CAFs). In response to intrinsic and extrinsic stressors, CAFs reorganize the TME enabling metastasis, therapeutic resistance, dormancy and reactivation by secreting cellular and acellular factors. In this review, we summarize the recent discoveries on CAF-mediated cancer progression with a particular focus on fibroblast heterogeneity and plasticity.

Stromal changes in the aged lung induce an emergence from melanoma dormancy.

Disseminated cancer cells from primary tumours can seed in distal tissues, but may take several years to form overt metastases, a phenomenon that is termed tumour dormancy. Despite its importance in metastasis and residual disease, few studies have been able to successfully characterize dormancy within melanoma. Here we show that the aged lung microenvironment facilitates a permissive niche for efficient outgrowth of dormant disseminated cancer cells-in contrast to the aged skin, in which age-related changes suppress melanoma growth but drive dissemination. These microenvironmental complexities can be explained by the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state1-3. It was previously shown that dermal fibroblasts promote phenotype switching in melanoma during ageing4-8. We now identify WNT5A as an activator of dormancy in melanoma disseminated cancer cells within the lung, which initially enables the efficient dissemination and seeding of melanoma cells in metastatic niches. Age-induced reprogramming of lung fibroblasts increases their secretion of the soluble WNT antagonist sFRP1, which inhibits WNT5A in melanoma cells and thereby enables efficient metastatic outgrowth. We also identify the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis within melanoma cells. Overall, we find that age-induced changes in distal metastatic microenvironments promote the efficient reactivation of dormant melanoma cells in the lung.

Reciprocal Regulation of BRN2 and NOTCH1/2 Signaling Synergistically Drives Melanoma Cell Migration and Invasion.

Phenotypic plasticity drives cancer progression, impacts treatment response, and is a major driver of therapeutic resistance. In melanoma, a regulatory axis between the MITF and BRN2 transcription factors has been reported to promote tumor heterogeneity by mediating switching between proliferative and invasive phenotypes, respectively. Despite strong evidence that subpopulations of cells that exhibit a BRN2high/MITFlow expression profile switch to a predominantly invasive phenotype, the mechanisms by which this switch is propagated and promotes invasion remain poorly defined. We have found that a reciprocal relationship between BRN2 and NOTCH1/2 signaling exists in melanoma cells in vitro, within patient datasets, and in in vivo primary and metastatic human tumors that bolsters acquisition of invasiveness. Working through the epigenetic modulator EZH2, the BRN2‒NOTCH1/2 axis is potentially a key mechanism by which the invasive phenotype is maintained. Given the emergence of agents targeting both EZH2 and NOTCH, understanding the mechanism through which BRN2 promotes heterogeneity may provide crucial biomarkers to predict treatment response to prevent metastasis.

Growth Hormone Stops Excessive Inflammation After Partial Hepatectomy, Allowing Liver Regeneration and Survival Through Induction of H2-Bl/HLA-G.

BACKGROUND AND AIMS: Growth hormone (GH) is important for liver regeneration after partial hepatectomy (PHx). We investigated this process in C57BL/6 mice that express different forms of the GH receptor (GHR) with deletions in key signaling domains. APPROACH AND RESULTS: PHx was performed on C57BL/6 mice lacking GHR (Ghr-/- ), disabled for all GH-dependent Janus kinase 2 signaling (Box1-/- ), or lacking only GH-dependent signal transducer and activator of transcription 5 (STAT5) signaling (Ghr391-/- ), and wild-type littermates. C57BL/6 Ghr-/- mice showed striking mortality within 48 hours after PHx, whereas Box1-/- or Ghr391-/- mice survived with normal liver regeneration. Ghr-/- mortality was associated with increased apoptosis and elevated natural killer/natural killer T cell and macrophage cell markers. We identified H2-Bl, a key immunotolerance protein, which is up-regulated by PHx through a GH-mediated, Janus kinase 2-independent, SRC family kinase-dependent pathway. GH treatment was confirmed to up-regulate expression of the human homolog of H2-Bl (human leukocyte antigen G [HLA-G]) in primary human hepatocytes and in the serum of GH-deficient patients. We find that injury-associated innate immune attack by natural killer/natural killer T cell and macrophage cells are instrumental in the failure of liver regeneration, and this can be overcome in Ghr-/- mice by adenoviral delivery of H2-Bl or by infusion of HLA-G protein. Further, H2-Bl knockdown in wild-type C57BL/6 mice showed elevated markers of inflammation after PHx, whereas Ghr-/- backcrossed on a strain with high endogenous H2-Bl expression showed a high rate of survival following PHx. CONCLUSIONS: GH induction of H2-Bl expression is crucial for reducing innate immune-mediated apoptosis and promoting survival after PHx in C57BL/6 mice. Treatment with HLA-G may lead to improved clinical outcomes following liver surgery or transplantation.

Myeloid-Derived Suppressor Cells Are a Major Source of Wnt5A in the Melanoma Microenvironment and Depend on Wnt5A for Full Suppressive Activity.

Metastatic dissemination remains a significant barrier to successful therapy for melanoma. Wnt5A is a potent driver of invasion in melanoma and is believed to be secreted from the tumor microenvironment (TME). Our data suggest that myeloid-derived suppressor cells (MDSC) in the TME are a major source of Wnt5A and are reliant upon Wnt5A for multiple actions. Knockdown of Wnt5A specifically in the myeloid cells demonstrated a clear decrease in Wnt5A expression within the TME in vivo as well as a decrease in intratumoral MDSC and regulatory T cell (Treg). Wnt5A knockdown also decreased the immunosuppressive nature of MDSC and decreased expression of TGFß1 and arginase 1. In the presence of Wnt5A-depleted MDSC, tumor-infiltrating lymphocytes expressed decreased PD-1 and LAG3, suggesting a less exhausted phenotype. Myeloid-specific Wnt5A knockdown also led to decreased lung metastasis. Tumor-infiltrating MDSC from control animals showed a strong positive correlation with Treg, which was completely ablated in animals with Wnt5A-negative MDSC. Overall, our data suggest that while MDSC contribute to an immunosuppressive and less immunogenic environment, they exhibit an additional function as the major source of Wnt5A in the TME. SIGNIFICANCE: These findings demonstrate that myeloid cells provide a major source of Wnt5A to facilitate metastatic potential in melanoma cells and rely on Wnt5A for their immunosuppressive function.

GHR signalling: Receptor activation and degradation mechanisms.

Growth hormone (GH) actions via initiating cell signalling through the GH receptor (GHR) are important for many physiological processes, in addition to its well-known role in regulating growth. The activation of JAK-STAT signalling by GH is well characterized, however knowledge on GH activation of SRC family kinases (SFKs) is still limited. In this review we summarise the collective knowledge on the activation, regulation, and downstream signalling of GHR. We highlight studies on GH activation of SFKs and the important outcome of this signalling pathway with a focus on the different degradation mechanisms that can regulate GHR availability since this is an area that warrants further study considering its role in tumour progression.

sFRP2 Supersedes VEGF as an Age-related Driver of Angiogenesis in Melanoma, Affecting Response to Anti-VEGF Therapy in Older Patients.

PURPOSE: Angiogenesis is thought to be critical for tumor metastasis. However, inhibiting angiogenesis using antibodies such as bevacizumab (Avastin), has had little impact on melanoma patient survival. We have demonstrated that both angiogenesis and metastasis are increased in older individuals, and therefore sought to investigate whether there was an age-related difference in response to bevacizumab, and if so, what the underlying mechanism could be. EXPERIMENTAL DESIGN: We analyzed data from the AVAST-M trial of 1,343 patients with melanoma treated with bevacizumab to determine whether there is an age-dependent response to bevacizumab. We also examined the age-dependent expression of VEGF and its cognate receptors in patients with melanoma, while using syngeneic melanoma animal models to target VEGF in young versus old mice. We also examined the age-related proangiogenic factor secreted frizzled-related protein 2 (sFRP2) and whether it could modulate response to anti-VEGF therapy. RESULTS: We show that older patients respond poorly to bevacizumab, whereas younger patients show improvement in both disease-free survival and overall survival. We find that targeting VEGF does not ablate angiogenesis in an aged mouse model, while sFRP2 promotes angiogenesis in vitro and in young mice. Targeting sFRP2 in aged mice successfully ablates angiogenesis, while the effects of targeting VEGF in young mice can be overcome by increasing sFRP2. CONCLUSIONS: VEGF is decreased during aging, thereby reducing response to bevacizumab. Despite the decrease in VEGF, angiogenesis is increased because of an increase in sFRP2 in the aged tumor microenvironment. These results stress the importance of considering age as a factor for designing targeted therapies.

Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning.

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F- essential thrombocythemia and primary myelofibrosis patients, respectively, have "canonical" MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other "noncanonical" MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

Growth hormone (GH) has an important function as an insulin antagonist with elevated insulin sensitivity evident in humans and mice lacking a functional GH receptor (GHR). We sought the molecular basis for this sensitivity by utilizing a panel of mice possessing specific deletions of GHR signaling pathways. Metabolic clamps and glucose homeostasis tests were undertaken in these obese adult C57BL/6 male mice, which indicated impaired hepatic gluconeogenesis. Insulin sensitivity and glucose disappearance rate were enhanced in muscle and adipose of mice lacking the ability to activate the signal transducer and activator of transcription (STAT)5 via the GHR (Ghr-391-/-) as for GHR-null (GHR-/-) mice. These changes were associated with a striking inhibition of hepatic glucose output associated with altered glycogen metabolism and elevated hepatic glycogen content during unfed state. The enhanced hepatic insulin sensitivity was associated with increased insulin receptor ß and insulin receptor substrate 1 activation along with activated downstream protein kinase B signaling cascades. Although phosphoenolpyruvate carboxykinase (Pck)-1 expression was unchanged, its inhibitory acetylation was elevated because of decreased sirtuin-2 expression, thereby promoting loss of PCK1. Loss of STAT5 signaling to defined chromatin immunoprecipitation targets would further increase lipogenesis, supporting hepatosteatosis while lowering glucose output. Finally, up-regulation of IL-15 expression in muscle, with increased secretion of adiponectin and fibroblast growth factor 1 from adipose tissue, is expected to promote insulin sensitivity.-Chhabra, Y., Nelson, C. N., Plescher, M., Barclay, J. L., Smith, A. G., Andrikopoulos, S., Mangiafico, S., Waxman, D. J., Brooks, A. J., Waters, M. J. Loss of growth hormone-mediated signal transducer and activator of transcription 5 (STAT5) signaling in mice results in insulin sensitivity with obesity.

BRN2, a POUerful driver of melanoma phenotype switching and metastasis.

The POU domain family of transcription factors play a central role in embryogenesis and are highly expressed in neural crest cells and the developing brain. BRN2 is a class III POU domain protein that is a key mediator of neuroendocrine and melanocytic development and differentiation. While BRN2 is a central regulator in numerous developmental programs, it has also emerged as a major player in the biology of tumourigenesis. In melanoma, BRN2 has been implicated as one of the master regulators of the acquisition of invasive behaviour within the phenotype switching model of progression. As a mediator of melanoma cell phenotype switching, it coordinates the transition to a dedifferentiated, slow cycling and highly motile cell type. Its inverse expression relationship with MITF is believed to mediate tumour progression and metastasis within this model. Recent evidence has now outlined a potential epigenetic switching mechanism in melanoma cells driven by BRN2 expression that induces melanoma cell invasion. We summarize the role of BRN2 in tumour cell dissemination and metastasis in melanoma, while also examining it as a potential metastatic regulator in other tumour models.

Growth hormone activated STAT5 is required for induction of beige fat in vivo.

OBJECTIVE: The anti-obesity actions of growth hormone (GH) led us to investigate if GH signaling is able to regulate beige/brite fat development of white adipose tissue (WAT). METHODS: We studied WAT in GHR-391 mice engineered to be unable to activate STAT5 in response to GH, in mice with adipose specific deletion of GHR, in GHR-/- mice and in bGH transgenic mice. QPCR, immunoblots and immunohistochemistry were used to characterize WAT. The in vivo effects of ß-3 adrenergic activation with CL-316,243 and that of FGF21 infusion were also studied. RESULTS: GHR-391 mice had lower surface temperature than WT, with deficiency in ß-oxidation and beiging transcripts including Ucp1. Oxidative phosphorylation complex subunit proteins were decreased dramatically in GHR-391 inguinal white adipose tissue (iWAT), but increased in bGH iWAT, as were proteins for beige/brown markers. In accord with its lack of ß-3 adrenergic receptors, iWAT of GHR-391 mice did not beige in response to administration of the ß-3 specific agonist CL-316,243 in contrast to WT mice. GHR-391 mice are deficient in FGF21, but unlike WT, infusion of the purified protein was without effect on extent of beiging. Finally, fat-specific deletion of the GHR replicated the loss of beiging associated transcripts. CONCLUSION: In addition to promoting lipolysis, our study suggests that GH is able to promote formation of beige adipose tissue through activation of STAT5 and induction of Adrb3. This sensitizes WAT to adrenergic input, and may contribute to the anti-obesity actions of GH.

Genetic variation in IRF4 expression modulates growth characteristics, tyrosinase expression and interferon-gamma response in melanocytic cells.

A SNP within intron4 of the interferon regulatory factor4 (IRF4) gene, rs12203592*C/T, has been independently associated with pigmentation and age-specific effects on naevus count in European-derived populations. We have characterized the cis-regulatory activity of this intronic region and using human foreskin-derived melanoblast strains, we have explored the correlation between IRF4 rs12203592 homozygous C/C and T/T genotypes with TYR enzyme activity, supporting its association with pigmentation traits. Further, higher IRF4 protein levels directed by the rs12203592*C allele were associated with increased basal proliferation but decreased cell viability following UVR, an etiological factor in melanoma development. Since UVR, and accompanying IFNγ-mediated inflammatory response, is associated with melanomagenesis, we evaluated its effects in the context of IRF4 status. Manipulation of IRF4 levels followed by IFNγ treatment revealed a subset of chemokines and immuno-evasive molecules that are sensitive to IRF4 expression level and genotype including CTLA4 and PD-L1.

NR4A2 Promotes DNA Double-strand Break Repair Upon Exposure to UVR.

Exposure of melanocytes to ultraviolet radiation (UVR) induces the formation of UV lesions that can produce deleterious effects in genomic DNA. Encounters of replication forks with unrepaired UV lesions can lead to several complex phenomena, such as the formation of DNA double-strand breaks (DSBs). The NR4A family of nuclear receptors are transcription factors that have been associated with mediating DNA repair functions downstream of the MC1R signaling pathway in melanocytes. In particular, emerging evidence shows that upon DNA damage, the NR4A2 receptor can translocate to sites of UV lesion by mechanisms requiring post-translational modifications within the N-terminal domain and at a serine residue in the DNA-binding domain at position 337. Following this, NR4A2 aids in DNA repair by facilitating chromatin relaxation, allowing accessibility for DNA repair machinery. Using A2058 and HT144 melanoma cells engineered to stably express wild-type or mutant forms of the NR4A2 proteins, we reveal that the expression of functional NR4A2 is associated with elevated cytoprotection against UVR. Conversely, knockdown of NR4A2 expression by siRNA results in a significant loss of cell viability after UV insult. By analyzing the kinetics of the ensuing 53BP1 and RAD51 foci following UV irradiation, we also reveal that the expression of mutant NR4A2 isoforms, lacking the ability to translocate, transactivate, or undergo phosphorylation, display compromised repair capacity.Implications: These data expand the understanding of the mechanism by which the NR4A2 nuclear receptor can facilitate DNA DSB repair. Mol Cancer Res; 15(9); 1184-96. ©2017 AACR.

NFIB Mediates BRN2 Driven Melanoma Cell Migration and Invasion Through Regulation of EZH2 and MITF.

While invasion and metastasis of tumour cells are the principle factor responsible for cancer related deaths, the mechanisms governing the process remain poorly defined. Moreover, phenotypic divergence of sub-populations of tumour cells is known to underpin alternative behaviors linked to tumour progression such as proliferation, survival and invasion. In the context of melanoma, heterogeneity between two transcription factors, BRN2 and MITF, has been associated with phenotypic switching between predominantly invasive and proliferative behaviors respectively. Epigenetic changes, in response to external cues, have been proposed to underpin this process, however the mechanism by which the phenotypic switch occurs is unclear. Here we report the identification of the NFIB transcription factor as a novel downstream effector of BRN2 function in melanoma cells linked to the migratory and invasive characteristics of these cells. Furthermore, the function of NFIB appears to drive an invasive phenotype through an epigenetic mechanism achieved via the upregulation of the polycomb group protein EZH2. A notable target of NFIB mediated up-regulation of EZH2 is decreased MITF expression, which further promotes a less proliferative, more invasive phenotype. Together our data reveal that NFIB has the ability to promote dynamic changes in the chromatin state of melanoma cells to facilitate migration, invasion and metastasis.

CRIM1 is necessary for coronary vascular endothelial cell development and homeostasis.

Endothelial cells form a critical component of the coronary vasculature, yet the factors regulating their development remain poorly defined. Here we reveal a novel role for the transmembrane protein CRIM1 in mediating cardiac endothelial cell development. In the absence of Crim1 in vivo, the coronary vasculature is malformed, the number of endothelial cells reduced, and the canonical BMP pathway dysregulated. Moreover, we reveal that CRIM1 can bind IGFs, and regulate IGF signalling within endothelial cells. Finally, loss of CRIM1 from human cardiac endothelial cells results in misregulation of endothelial genes, predicted by pathway analysis to be involved in an increased inflammatory response and cytolysis, reminiscent of endothelial cell dysfunction in cardiovascular disease pathogenesis. Collectively, these findings implicate CRIM1 in endothelial cell development and homeostasis in the coronary vasculature.