HILPDA promotes NASH-driven HCC development by restraining intracellular fatty acid flux in hypoxia.

BACKGROUND & AIMS: The prevalence of non-alcoholic steatohepatitis (NASH)-driven hepatocellular carcinoma (HCC) is rising rapidly, yet its underlying mechanisms remain unclear. Herein, we aim to determine the role of hypoxia-inducible lipid droplet associated protein (HILPDA)/hypoxia-inducible gene 2 (HIG2), a selective inhibitor of intracellular lipolysis, in NASH-driven HCC. METHODS: The clinical significance of HILPDA was assessed in human NASH-driven HCC specimens by immunohistochemistry and transcriptomics analyses. The oncogenic effect of HILPDA was assessed in human HCC cells and in 3D epithelial spheroids upon exposure to free fatty acids and either normoxia or hypoxia. Lipidomics profiling of wild-type and HILPDA knockout HCC cells was assessed via shotgun and targeted approaches. Wild-type (Hilpdafl/fl) and hepatocyte-specific Hilpda knockout (HilpdaΔHep) mice were fed a Western diet and high sugar in drinking water while receiving carbon tetrachloride to induce NASH-driven HCC. RESULTS: In patients with NASH-driven HCC, upregulated HILPDA expression is strongly associated with poor survival. In oxygen-deprived and lipid-loaded culture conditions, HILPDA promotes viability of human hepatoma cells and growth of 3D epithelial spheroids. Lack of HILPDA triggered flux of polyunsaturated fatty acids to membrane phospholipids and of saturated fatty acids to ceramide synthesis, exacerbating lipid peroxidation and apoptosis in hypoxia. The apoptosis induced by HILPDA deficiency was reversed by pharmacological inhibition of ceramide synthesis. In our experimental mouse model of NASH-driven HCC, HilpdaΔHep exhibited reduced hepatic steatosis and tumorigenesis but increased oxidative stress in the liver. Single-cell analysis supports a dual role of hepatic HILPDA in protecting HCC cells and facilitating the establishment of a pro-tumorigenic immune microenvironment in NASH. CONCLUSIONS: Hepatic HILPDA is a pivotal oncometabolic factor in the NASH liver microenvironment and represents a potential novel therapeutic target. IMPACT AND IMPLICATIONS: Non-alcoholic steatohepatitis (NASH, chronic metabolic liver disease caused by buildup of fat, inflammation and damage in the liver) is emerging as the leading risk factor and the fastest growing cause of hepatocellular carcinoma (HCC), the most common form of liver cancer. While curative therapeutic options exist for HCC, it frequently presents at a late stage when such options are no longer effective and only systemic therapies are available. However, systemic therapies are still associated with poor efficacy and some side effects. In addition, no approved drugs are available for NASH. Therefore, understanding the underlying metabolic alterations occurring during NASH-driven HCC is key to identifying new cancer treatments that target the unique metabolic needs of cancer cells.

Interleukin-6 promotes pancreatic cancer cell migration by rapidly activating the small GTPase CDC42.

Inflammation is a major driver of tumor progression and metastasis, although the mechanisms by which proinflammatory cytokines drive metastatic invasion are unknown. Interleukin-6 (IL-6) is a potent proinflammatory cytokine that is elevated in individuals with pancreatic cancer (PDAC), is required for PDAC progression in mice, and increases tumor cell invasion in vitro Here, we provide insights into the mechanisms by which IL-6 activates tumor cell invasion. We found that IL-6 stimulation rapidly and robustly activates the small GTPase cell division cycle 42 (CDC42) in human PDAC cells and promotes the formation of premigratory filopodia. The CDC42 activation was required for IL-6-induced invasion as blocking CDC42 activity rendered the cells insensitive to IL-6's proinvasive effects. Loss of Janus kinase 2 (JAK2) or signal transducer and activator of transcription 3 (STAT3) prevented IL-6-mediated CDC42 activation, indicating that IL-6 activates CDC42 through both JAK2 and STAT3. However, the rapid activation of CDC42 suggested that this activation may be distinct from canonical STAT3-mediated transcriptional activation. Importantly, we observed an interaction between STAT3 and IQ motif-containing GTPase-activating protein 1 (IQGAP1), a scaffolding platform that binds CDC42. STAT3 colocalized with CDC42 and IQGAP1 at the plasma membrane, suggesting cross-talk between IL-6-mediated STAT3 signaling and CDC42 activation. These results suggest that IL-6 promotes metastatic invasion, at least partially, through CDC42 and that, along with its pleiotropic effects on tumor growth and progression, IL-6 signaling also activates proinvasive GTPase signaling, priming tumor cells for metastatic invasion.

Integrated mate-pair and RNA sequencing identifies novel, targetable gene fusions in peripheral T-cell lymphoma.

Peripheral T-cell lymphomas (PTCLs) represent a heterogeneous group of T-cell malignancies that generally demonstrate aggressive clinical behavior, often are refractory to standard therapy, and remain significantly understudied. The most common World Health Organization subtype is PTCL, not otherwise specified (NOS), essentially a "wastebasket" category because of inadequate understanding to assign cases to a more specific diagnostic entity. Identification of novel fusion genes has contributed significantly to improving the classification, biologic understanding, and therapeutic targeting of PTCLs. Here, we integrated mate-pair DNA and RNA next-generation sequencing to identify chromosomal rearrangements encoding expressed fusion transcripts in PTCL, NOS. Two of 11 cases had novel fusions involving VAV1, encoding a truncated form of the VAV1 guanine nucleotide exchange factor important in T-cell receptor signaling. Fluorescence in situ hybridization studies identified VAV1 rearrangements in 10 of 148 PTCLs (7%). These were observed exclusively in PTCL, NOS (11%) and anaplastic large cell lymphoma (11%). In vitro, ectopic expression of a VAV1 fusion promoted cell growth and migration in a RAC1-dependent manner. This growth was inhibited by azathioprine, a clinically available RAC1 inhibitor. We also identified novel kinase gene fusions, ITK-FER and IKZF2-ERBB4, as candidate therapeutic targets that show similarities to known recurrent oncogenic ITK-SYK fusions and ERBB4 transcript variants in PTCLs, respectively. Additional novel and potentially clinically relevant fusions also were discovered. Together, these findings identify VAV1 fusions as recurrent and targetable events in PTCLs and highlight the potential for clinical sequencing to guide individualized therapy approaches for this group of aggressive malignancies.

The Cell Biology of Metastatic Invasion in Pancreatic Cancer: Updates and Mechanistic Insights.

Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related mortality worldwide. This is largely due to the lack of routine screening protocols, an absence of symptoms in early-stage disease leading to late detection, and a paucity of effective treatment options. Critically, the majority of patients either present with metastatic disease or rapidly develop metastatic disease. Thus, there is an urgent need to deepen our understanding of metastasis in PDAC. During metastasis, tumor cells escape from the primary tumor, enter the circulation, and travel to a distant site to form a secondary tumor. In order to accomplish this relatively rare event, tumor cells develop an enhanced ability to detach from the primary tumor, migrate into the surrounding matrix, and invade across the basement membrane. In addition, cancer cells interact with the various cell types and matrix proteins that comprise the tumor microenvironment, with some of these factors working to promote metastasis and others working to suppress it. In PDAC, many of these processes are not well understood. The purpose of this review is to highlight recent advances in the cell biology of the early steps of the metastatic cascade in pancreatic cancer. Specifically, we will examine the regulation of epithelial-to-mesenchymal transition (EMT) in PDAC and its requirement for metastasis, summarize our understanding of how PDAC cells invade and degrade the surrounding matrix, and discuss how migration and adhesion dynamics are regulated in PDAC to optimize cancer cell motility. In addition, the role of the tumor microenvironment in PDAC will also be discussed for each of these invasive processes.

FSCN1 and epithelial mesenchymal transformation transcription factor expression in human pancreatic intraepithelial neoplasia and ductal adenocarcinoma.

BACKGROUND: The actin regulatory protein fascin (FSCN1) and epithelial mesenchymal transition (EMT) transcription factor (TF) SLUG/SNAI2 have been shown to be expressed in PDAC and its precursor lesions (pancreatic intraepithelial neoplasia (PanIN), graded 1-3) in in vitro and murine in vivo studies. Our aim was to investigate the expression of FSCN1 and EMT-TFs and their association with survival in human PanIN and PDAC. METHODS: Expression was investigated in silico using TCGA PanCancer Atlas data (177 PDAC samples with mRNA data) and immunohistochemical staining of a tissue microarray (TMA) (59 PDAC patients). RESULTS: High FSCN1 expression was associated with poorer overall survival (p = 0.02) in the TCGA data. EMT-TF expression was not associated with survival, however FSCN1 expression correlated with that of the EMT-TFs SLUG/SNAI2 (rho = 0.49, p < 0.001) and TWIST1 (rho = 0.52, p < 0.001). TMA IHC showed low expression of SNAI2 and TWIST1 in normal ductal epithelium, while FSCN1 was not expressed. SNAI2 increased slightly in PanIN1-2, then decreased in higher grade lesions. TWIST1 increased in PanIN2-3 and was retained in PDAC. FSCN1 was increasingly expressed from PanIN2 onwards. SNAI2 and TWIST1 expression positively correlated in all grades of PanIN and PDAC (rho = 0.52, p < 0.001). FSCN1 correlated positively with SNAI2 in PanIN1 (rho = 0.56, p < 0.01). CONCLUSIONS: Increased expression of EMT-TFs in low-grade PanIN followed by FSCN1 in PanIN3 and PDAC suggests EMT-TFs may trigger FSCN1 expression and are potential early diagnostic markers. FSCN1 expression correlated with overall survival in PDAC and may have value as a prognostic marker.

Ectopic expression of DOCK8 regulates lysosome-mediated pancreatic tumor cell invasion.

Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor growth and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) dedicator of cytokinesis 8 (DOCK8) on lysosomes. Surprisingly, DOCK8 is aberrantly expressed in a subset of PDAC, where it promotes cell invasion in vitro and in vivo. DOCK8 associates with lysosomes and regulates lysosomal morphology and motility, with loss of DOCK8 leading to increased lysosome size. DOCK8 promotes actin polymerization at the surface of lysosomes while also increasing the proteolytic activity of the lysosomal protease cathepsin B. Critically, depletion of DOCK8 significantly reduces cathepsin-dependent extracellular matrix degradation and impairs the invasive capacity of PDAC cells. These findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation and invasion in pancreatic cancer cells.

Myotubularin regulates Akt-dependent survival signaling via phosphatidylinositol 3-phosphate.

Myotubularin is a 3-phosphoinositide phosphatase that is mutated in X-linked myotubular myopathy, a severe neonatal disorder in which skeletal muscle development and/or regeneration is impaired. In this report we provide evidence that siRNA-mediated silencing of myotubularin expression markedly inhibits growth factor-stimulated Akt phosphorylation, leading to activation of caspase-dependent pro-apoptotic signaling in HeLa cells and primary human skeletal muscle myotubes. Myotubularin silencing also inhibits Akt-dependent signaling through the mammalian target of rapamycin complex 1 as assessed by p70 S6-kinase and 4E-BP1 phosphorylation. Similarly, phosphorylation of FoxO transcription factors is also significantly reduced in myotubularin-deficient cells. Our data further suggest that inhibition of Akt activation and downstream survival signaling in myotubularin-deficient cells is caused by accumulation of the MTMR substrate lipid phosphatidylinositol 3-phosphate generated from the type II phosphatidylinositol 3-kinase PIK3C2B. Our findings are significant because they suggest that myotubularin regulates Akt activation via a cellular pool of phosphatidylinositol 3-phosphate that is distinct from that generated by the type III phosphatidylinositol 3-kinase hVps34. Because impaired Akt signaling has been tightly linked to skeletal muscle atrophy, we hypothesize that loss of Akt-dependent growth/survival cues due to impaired myotubularin function may be a critical factor underlying the severe skeletal muscle atrophy characteristic of muscle fibers in patients with X-linked myotubular myopathy.

Anticachectic regulator analysis reveals Perp-dependent antitumorigenic properties of 3-methyladenine in pancreatic cancer.

Approximately 80% of pancreatic cancer patients suffer from cachexia, and one-third die due to cachexia-related complications such as respiratory failure and cardiac arrest. Although there has been considerable research into cachexia mechanisms and interventions, there are, to date, no FDA-approved therapies. A major contributing factor for the lack of therapy options could be the failure of animal models to accurately recapitulate the human condition. In this study, we generated an aged model of pancreatic cancer cachexia to compare cachexia progression in young versus aged tumor-bearing mice. Comparative skeletal muscle transcriptome analyses identified 3-methyladenine (3-MA) as a candidate antiwasting compound. In vitro analyses confirmed antiwasting capacity, while in vivo analysis revealed potent antitumor effects. Transcriptome analyses of 3-MA-treated tumor cells implicated Perp as a 3-MA target gene. We subsequently (a) observed significantly higher expression of Perp in cancer cell lines compared with control cells, (b) noted a survival disadvantage associated with elevated Perp, and (c) found that 3-MA-associated Perp reduction inhibited tumor cell growth. Finally, we have provided in vivo evidence that survival benefits conferred by 3-MA administration are independent of its effect on tumor progression. Taken together, we report a mechanism linking 3-MA to Perp inhibition, and we further implicate Perp as a tumor-promoting factor in pancreatic cancer.

Distinct forms of the actin cross-linking protein α-actinin support macropinosome internalization and trafficking.

The α-actinin family of actin cross-linking proteins have been implicated in driving tumor cell metastasis through regulation of the actin cytoskeleton; however, there has been little investigation into whether these proteins can influence tumor cell growth. We demonstrate that α-actinin 1 and 4 are essential for nutrient uptake through the process of macropinocytosis in pancreatic ductal adenocarcinoma (PDAC) cells, and inhibition of these proteins decreases tumor cell survival in the presence of extracellular protein. The α-actinin proteins play essential roles throughout the macropinocytic process, where α-actinin 4 stabilizes the actin cytoskeleton on the plasma membrane to drive membrane ruffling and macropinosome internalization and α-actinin 1 localizes to actin tails on macropinosomes to facilitate trafficking to the lysosome for degradation. In addition to tumor cell growth, we also observe that the α-actinin proteins can influence uptake of chemotherapeutics and extracellular matrix proteins through macropinocytosis, suggesting that the α-actinin proteins can regulate multiple tumor cell properties through this endocytic process. In summary, these data demonstrate a critical role for the α-actinin isoforms in tumor cell macropinocytosis, thereby affecting the growth and invasive potential of PDAC tumors.

Synergistic metalloproteinase-based remodeling of matrix by pancreatic tumor and stromal cells.

The process by which tumor cells mechanically invade through the surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. Matrix metalloproteinase (MMP)-mediated extracellular matrix (ECM) degradation plays an important role in this invasive process. Defining the contribution and interaction between these MMPs during invasion remains a key interest in the development of targeted anti-metastatic therapies. In this study we have utilized multiple different stromal fibroblasts and tumor cells to define the relative contributions between cancer cells and stromal cells during MMP-dependent matrix remodeling and pancreatic (PDAC) tumor cell invasion. We find that tumor cells co-cultured with the conditioned medium from stromal fibroblasts exhibited a substantial increase in invadopodial-based matrix degradation and transwell invasion. This increase is dependent on pro-MMP2 expressed and secreted by stromal fibroblasts. Further, the pro-MMP2 from the stromal fibroblasts is activated by MT1-MMP expressed on the tumor cells. Depletion of MT1-MMP, the known activator of MMP2, in tumor cells largely blocked matrix remodeling, even in the presence of stromal cell medium. In summary, these findings implicate an important interplay between MT1-MMP from tumor cells and MMP2 from fibroblasts as a key component for ECM remodeling and invasion.

GAS7 Deficiency Promotes Metastasis in MYCN-Driven Neuroblastoma.

One of the greatest barriers to curative treatment of neuroblastoma is its frequent metastatic outgrowth prior to diagnosis, especially in cases driven by amplification of the MYCN oncogene. However, only a limited number of regulatory proteins that contribute to this complex MYCN-mediated process have been elucidated. Here we show that the growth arrest-specific 7 (GAS7) gene, located at chromosome band 17p13.1, is preferentially deleted in high-risk MYCN-driven neuroblastoma. GAS7 expression was also suppressed in MYCN-amplified neuroblastoma lacking 17p deletion. GAS7 deficiency led to accelerated metastasis in both zebrafish and mammalian models of neuroblastoma with overexpression or amplification of MYCN. Analysis of expression profiles and the ultrastructure of zebrafish neuroblastoma tumors with MYCN overexpression identified that GAS7 deficiency led to (i) downregulation of genes involved in cell-cell interaction, (ii) loss of contact among tumor cells as critical determinants of accelerated metastasis, and (iii) increased levels of MYCN protein. These results provide the first genetic evidence that GAS7 depletion is a critical early step in the cascade of events culminating in neuroblastoma metastasis in the context of MYCN overexpression. SIGNIFICANCE: Heterozygous deletion or MYCN-mediated repression of GAS7 in neuroblastoma releases an important brake on tumor cell dispersion and migration to distant sites, providing a novel mechanism underlying tumor metastasis in MYCN-driven neuroblastoma.See related commentary by Menard, p. 2815.

Ras regulates assembly of mitogenic signalling complexes through the effector protein IMP.

The signal transduction cascade comprising Raf, mitogen-activated protein (MAP) kinase kinase (MEK) and MAP kinase is a Ras effector pathway that mediates diverse cellular responses to environmental cues and contributes to Ras-dependent oncogenic transformation. Here we report that the Ras effector protein Impedes Mitogenic signal Propagation (IMP) modulates sensitivity of the MAP kinase cascade to stimulus-dependent activation by limiting functional assembly of the core enzymatic components through the inactivation of KSR, a scaffold/adaptor protein that couples activated Raf to its substrate MEK. IMP is a Ras-responsive E3 ubiquitin ligase that, on activation of Ras, is modified by auto-polyubiquitination, which releases the inhibition of Raf-MEK complex formation. Thus, Ras activates the MAP kinase cascade through simultaneous dual effector interactions: induction of Raf kinase activity and derepression of Raf-MEK complex formation. IMP depletion results in increased stimulus-dependent MEK activation without alterations in the timing or duration of the response. These observations suggest that IMP functions as a threshold modulator, controlling sensitivity of the cascade to stimulus and providing a mechanism to allow adaptive behaviour of the cascade in chronic or complex signalling environments.

KRAS Controls Pancreatic Cancer Cell Lipid Metabolism and Invasive Potential through the Lipase HSL.

Oncogene-induced metabolic reprogramming is a hallmark of pancreatic cancer (PDAC), yet the metabolic drivers of metastasis are unclear. In PDAC, obesity and excess fatty acids accelerate tumor growth and increase metastasis. Here, we report that excess lipids, stored in organelles called lipid droplets (LD), are a key resource to fuel the energy-intensive process of metastasis. The oncogene KRAS controlled the storage and utilization of LD through regulation of hormone-sensitive lipase (HSL), which was downregulated in human PDAC. Disruption of the KRAS-HSL axis reduced lipid storage, reprogrammed tumor cell metabolism, and inhibited invasive migration in vitro and metastasis in vivo. Finally, microscopy-based metabolic analysis revealed that migratory cells selectively utilize oxidative metabolism during the process of migration to metabolize stored lipids and fuel invasive migration. Taken together, these results reveal a mechanism that can be targeted to attenuate PDAC metastasis. SIGNIFICANCE: KRAS-dependent regulation of HSL biases cells towards lipid storage for subsequent utilization during invasion of pancreatic cancer cells, representing a potential target for therapeutic intervention.See related commentary by Man et al., p. 4886.

Dynamin 2 interacts with α-actinin 4 to drive tumor cell invasion.

The large GTPase Dynamin 2 (Dyn2) is known to increase the invasiveness of pancreatic cancer tumor cells, but the mechanisms by which Dyn2 regulates changes in the actin cytoskeleton to drive cell migration are still unclear. Here we report that a direct interaction between Dyn2 and the actin-bundling protein alpha-actinin (α-actinin) 4 is critical for tumor cell migration and remodeling of the extracellular matrix in pancreatic ductal adenocarcinoma (PDAC) cells. The direct interaction is mediated through the C-terminal tails of both Dyn2 and α-actinin 4, and these proteins interact at invasive structures at the plasma membrane. While Dyn2 binds directly to both α-actinin 1 and α-actinin 4, only the interaction with α-actinin 4 is required to promote tumor cell invasion. Specific disruption of the Dyn2-α-actinin 4 interaction blocks the ability of PDAC cells to migrate in either two dimensions or invade through extracellular matrix as a result of impaired invadopodia stability. Analysis of human PDAC tumor tissue additionally reveals that elevated α-actinin 4 or Dyn2 expression are predictive of poor survival. Overall, these data demonstrate that Dyn2 regulates cytoskeletal dynamics, in part, by interacting with the actin-binding protein α-actinin 4 during tumor cell invasion.

The molecular scaffold kinase suppressor of Ras 1 is a modifier of RasV12-induced and replicative senescence.

In primary mouse embryo fibroblasts (MEFs), oncogenic Ras induces growth arrest via Raf/MEK/extracellular signal-regulated kinase (ERK)-mediated activation of the p19ARF/p53 and INK4/Rb tumor suppressor pathways. Ablation of these same pathways causes spontaneous immortalization in MEFs, and oncogenic transformation by Ras requires ablation of one or both of these pathways. We show that Kinase Suppressor of Ras 1 (KSR1), a molecular scaffold for the Raf/MEK/ERK cascade, is necessary for RasV12-induced senescence, and its disruption enhances primary MEF immortalization. RasV12 failed to induce p53, p19ARF, p16INK4a, and p15INK4b expression in KSR1-/- MEFs and increased proliferation instead of causing growth arrest. Reintroduction of wild-type KSR1, but not a mutated KSR1 construct unable to bind activated ERK, rescued RasV12-induced senescence. On continuous culture, deletion of KSR1 accelerated the establishment of spontaneously immortalized cultures and increased the proportion of cultures escaping replicative crisis. Despite enhancing escape from both RasV12-induced and replicative senescence, however, both primary and immortalized KSR1-/- MEFs are completely resistant to RasV12-induced transformation. These data show that escape from senescence is not necessarily a precursor for oncogenic transformation. Furthermore, these data indicate that KSR1 is a member of a unique class of proteins whose deletion blocks both senescence and transformation.

Genetic alterations affecting GTPases and T-cell receptor signaling in peripheral T-cell lymphomas.

Peripheral T-cell lymphomas (PTCLs) are rare, heterogeneous tumors with poor response to standard therapy and few targeted treatments available. The identification of mutations in the T-cell receptor (TCR) signaling pathway that either directly or indirectly affect Ras- and Rho-family GTPases is an emerging theme across PTCL subtypes. This review summarizes the role of GTPases in TCR signaling and highlights the constellation of mutations in this pathway among PTCLs. In particular, focus is given to the functional impact of the mutations and opportunities for targeted therapy. These mutations include activating mutations and gene fusions involving the guanine nucleotide exchange factor, VAV1, as well as activating and dominant negative mutations in the GTPases KRAS and RHOA, respectively. In addition to mutations directly affecting the GTPase pathway, TCR signaling mutations indirectly affecting Ras- and Rho-family GTPases involving genes such as CD28, FYN, LCK, and PLCG1 are also reviewed.

Pancreatic tumor cell metastasis is restricted by MT1-MMP binding protein MTCBP-1.

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1-expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

The molecular scaffold kinase suppressor of Ras 1 (KSR1) regulates adipogenesis.

Mitogen-activated protein kinase pathways are implicated in the regulation of cell differentiation, although their precise roles in many differentiation programs remain elusive. The Raf/MEK/extracellular signal-regulated kinase (ERK) kinase cascade has been proposed to both promote and inhibit adipogenesis. Here, we titrate expression of the molecular scaffold kinase suppressor of Ras 1 (KSR1) to regulate signaling through the Raf/MEK/ERK/p90 ribosomal S6 kinase (RSK) kinase cascade and show how it determines adipogenic potential. Deletion of KSR1 prevents adipogenesis in vitro, which can be rescued by introduction of low levels of KSR1. Appropriate levels of KSR1 coordinate ERK and RSK activation with C/EBPbeta synthesis leading to the phosphorylation and stabilization of C/EBPbeta at the precise moment it is required within the adipogenic program. Elevated levels of KSR1 expression, previously shown to enhance cell proliferation, promote high, sustained ERK activation that phosphorylates and inhibits peroxisome proliferator-activated receptor gamma, inhibiting adipogenesis. Titration of KSR1 expression reveals how a molecular scaffold can modulate the intensity and duration of signaling emanating from a single pathway to dictate cell fate.

Stromal fibroblasts facilitate cancer cell invasion by a novel invadopodia-independent matrix degradation process.

Metastatic invasion of tumors into peripheral tissues is known to rely upon protease-mediated degradation of the surrounding stroma. This remodeling process uses complex, actin-based, specializations of the plasma membrane termed invadopodia that act both to sequester and release matrix metalloproteinases. Here we report that cells of mesenchymal origin, including tumor-associated fibroblasts, degrade substantial amounts of surrounding matrix by a mechanism independent of conventional invadopodia. These degradative sites lack the punctate shape of conventional invadopodia to spread along the cell base and are reticular and/or fibrous in character. In marked contrast to invadopodia, this degradation does not require the action of Src kinase, Cdc42 or Dyn2. Rather, inhibition of Dyn2 causes a marked upregulation of stromal matrix degradation. Further, expression and activity of matrix metalloproteinases are differentially regulated between tumor cells and stromal fibroblasts. This matrix remodeling by fibroblasts increases the invasive capacity of tumor cells, thereby illustrating how the tumor microenvironment can contribute to metastasis. These findings provide evidence for a novel matrix remodeling process conducted by stromal fibroblasts that is substantially more effective than conventional invadopodia, distinct in structural organization and regulated by disparate molecular mechanisms.