Deep Learning Reveals Cancer Metastasis and Therapeutic Antibody Targeting in the Entire Body.

Reliable detection of disseminated tumor cells and of the biodistribution of tumor-targeting therapeutic antibodies within the entire body has long been needed to better understand and treat cancer metastasis. Here, we developed an integrated pipeline for automated quantification of cancer metastases and therapeutic antibody targeting, named DeepMACT. First, we enhanced the fluorescent signal of cancer cells more than 100-fold by applying the vDISCO method to image metastasis in transparent mice. Second, we developed deep learning algorithms for automated quantification of metastases with an accuracy matching human expert manual annotation. Deep learning-based quantification in 5 different metastatic cancer models including breast, lung, and pancreatic cancer with distinct organotropisms allowed us to systematically analyze features such as size, shape, spatial distribution, and the degree to which metastases are targeted by a therapeutic monoclonal antibody in entire mice. DeepMACT can thus considerably improve the discovery of effective antibody-based therapeutics at the pre-clinical stage. VIDEO ABSTRACT.

DNA methylation-based classification of central nervous system tumours.

Accurate pathological diagnosis is crucial for optimal management of patients with cancer. For the approximately 100 known tumour types of the central nervous system, standardization of the diagnostic process has been shown to be particularly challenging-with substantial inter-observer variability in the histopathological diagnosis of many tumour types. Here we present a comprehensive approach for the DNA methylation-based classification of central nervous system tumours across all entities and age groups, and demonstrate its application in a routine diagnostic setting. We show that the availability of this method may have a substantial impact on diagnostic precision compared to standard methods, resulting in a change of diagnosis in up to 12% of prospective cases. For broader accessibility, we have designed a free online classifier tool, the use of which does not require any additional onsite data processing. Our results provide a blueprint for the generation of machine-learning-based tumour classifiers across other cancer entities, with the potential to fundamentally transform tumour pathology.

Spatiotemporally controlled induction of gene expression in vivo allows tracking the fate of tumor cells that traffic through the lymphatics.

Metastasis is a multistep process, during which circulating tumor cells traffic through diverse anatomical locations. Stable inducible marking of tumor cells in a manner that is tightly spatially and temporally controlled would allow tracking the contribution of cells passing through specific locations to metastatic dissemination. For example, tumor cells enter the lymphatic system and can form metastases in regional lymph nodes, but the relative contribution of tumor cells that traffic through the lymphatic system to the formation of distant metastases remains controversial. Here, we developed a novel genetic switch based on mild transient warming (TW) that allows cells to be marked in a defined spatiotemporal manner in vivo. Prior to warming, cells express only EGFP. Upon TW, the EGFP gene is excised and expression of mCherry is permanently turned on. We employed this system in an experimental pancreatic cancer model and used localized TW to induce the genetic switch in tumor cells trafficking through tumor-draining lymph nodes. Thereby we found that tumor cells disseminating via the lymphatics make a major contribution to the seeding of lung metastases. The inducible genetic marking system we have developed is a powerful tool for the tracking of metastasizing cells in vivo.

Design, synthesis and biological evaluation of fused naphthofuro[3,2-c] quinoline-6,7,12-triones and pyrano[3,2-c]quinoline-6,7,8,13-tetraones derivatives as ERK inhibitors with efficacy in BRAF-mutant melanoma.

Approximately 60% of human cancers exhibit enhanced activity of ERK1 and ERK2, reflecting their multiple roles in tumor initiation and progression. Acquired drug resistance, especially mechanisms associated with the reactivation of the MAPK (RAF/MEK/ERK) pathway represent a major challenge to current treatments of melanoma and several other cancers. Recently, targeting ERK has evolved as a potentially attractive strategy to overcome this resistance. Herein, we report the design and synthesis of novel series of fused naphthofuro[3,2-c]quinoline-6,7,12-triones 3a-f and pyrano[3,2-c]quinoline-6,7,8,13-tetraones 5a,b and 6, as potential ERK inhibitors. New inhibitors were synthesized and identified by different spectroscopic techniques and X-ray crystallography. They were evaluated for their ability to inhibit ERK1/2 in an in vitro radioactive kinase assay. 3b and 6 inhibited ERK1 with IC50s of 0.5 and 0.19 µM, and inhibited ERK2 with IC50s of 0.6 and 0.16 µM respectively. Kinetic mechanism studies revealed that the inhibitors are ATP-competitive inhibitors where 6 inhibited ERK2 with a Ki of 0.09 µM. Six of the new inhibitors were tested for their in vitro anticancer activity against the NCI-60 panel of tumor cell lines. Compound 3b and 6 were the most potent against most of the human tumor cell lines tested. Moreover, 3b and 6 inhibited the proliferation of the BRAF mutant A375 melanoma cells with IC50s of 3.7 and 0.13 µM, respectively. In addition, they suppressed anchorage-dependent colony formation. Treatment of the A375 cell line with 3b and 6 inhibited the phosphorylation of ERK substrates p-90RSK and ELK-1 and induced apoptosis in a dose dependent manner. Finally, a molecular docking study showed the potential binding mode of 3b and 6 within the ATP catalytic binding site of ERK2.

Implications of Oxygen Homeostasis for Tumor Biology and Treatment.

Tumors serve as a prototype system to study the role of the hypoxic microenvironment and gain insight in the regulation oxygen homeostasis. A series of biochemical and cell biological studies have significantly extended our knowledge of how tumor cells activate key regulatory mechanisms of oxygen homeostasis not only to adapt to the hostile tumor microenvironment but also to acquire a more aggressive tumor phenotype. Reduced oxygen levels and tumor-specific genetic alterations synergistically drive tumor progression by activating a key transcriptional system, the hypoxia inducible factors (HIFs). HIFs trigger a set of adaptive responses commonly associated with tumor malignancy including tumor angiogenesis, a shift in metabolism, proliferation, invasion, and metastasis. We and others could demonstrate that cancer stem cells are controlled by HIFs within a hypoxic niche, establishing an intriguing link between the well known function of hypoxia in tumor growth and stem cell biology. Additionally, HIF activation potentially conveys resistance to current tumor therapies including the evasive resistance phenotype observed after anti-angiogenic treatment. Together, these findings provide strong evidence that activation of the HIF system is a decisive step in cancer progression that critically shapes therapy response and clinical outcome. Recent insight into the precise mechanisms of oxygen sensing and signalling has offered new promising and potentially selective strategies to counteract this crucial pathway.

Rapidly progressive hypertrophic cardiomyopathy in an infant with Noonan syndrome with multiple lentigines: palliative treatment with a rapamycin analog.

Noonan syndrome with multiple lentigines (NSML) frequently manifests with hypertrophic cardiomyopathy (HCM). Recently, it was demonstrated that mTOR inhibition reverses HCM in NSML mice. We report for the first time on the effects of treatment with a rapamycin analog in an infant with LS and malignant HCM. In the boy, progressive HCM was diagnosed during the first week of life and a diagnosis of NSML was established at age 20 weeks by showing a heterozygous Q510E mutation in PTPN11. Immunoblotting with antibodies against pERK, pAkt, and pS6RP in fibroblasts demonstrated enhanced Akt/mTOR pathway activity. Because of the patient's critical condition, everolimus therapy was started at age 24 weeks and continued until heart transplantation at age 36 weeks. Prior to surgery, heart failure improved from NYHA stage IV to II and brain natriuretic peptide values decreased from 9,600 to <1,000 pg/ml, but no reversal of cardiac hypertrophy was observed. Examination of the explanted heart revealed severe hypertrophy and myofiber disarray with extensive perivascular fibrosis. These findings provide evidence that Akt/mTOR activity is enhanced in NSML with HCM and suggest that rapamycin treatment could principally be feasible for infantile NSML. The preliminary experiences made in this single patient indicate that therapy should start early to prevent irreversible cardiac remodelling.

The cancer stem cell niche(s): the crosstalk between glioma stem cells and their microenvironment.

BACKGROUND: The initiation and progression of various types of tumors, including glioma, are driven by a population of cells with stem cell properties. Glioma stem cells (GSCs) are located in specialized microenvironments (niches) within tumors. These niches represent the hallmarks of malignant gliomas (vascular proliferations, hypoxia/necrosis) and bear analogy to the microenvironments in which physiological stem cells in the brain are found. SCOPE OF THE REVIEW: Here we review the progress that has been made towards uncovering the function of the perivascular and the hypoxic niche and the molecular pathways that control the properties of GSCs within them. We propose models of how the different niches and GSC pools in them interact with each other. MAJOR CONCLUSIONS: GSCs are not merely passive residents of their niches, but actively contribute to the shaping of the niches through a complex crosstalk with different components of the microenvironment. For example, GSCs play a dominant role in promoting new blood vessel formation through a variety of mechanisms, including the hypoxia dependent stimulation of angiogenesis, recruitment of endothelial progenitor cells and direct transdifferentiation into endothelial cells. Recent work has also revealed that GSCs can recruit and modulate the function of various immune cells to suppress anti-tumor immune responses and to foster tumor-promoting inflammation, which in turn could support the maintenance of GSCs. GENERAL SIGNIFICANCE: These findings underscore the central role of the GSC microenvironment in driving glioma progression making the GSC niche a prime therapeutic target for the design of therapies aimed at eradicating GSCs. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Increased Circulating Osteopontin Levels Promote Primary Tumour Growth, but Do Not Induce Metastasis in Melanoma.

Osteopontin (OPN) is a phosphoprotein with diverse functions in various physiological and pathological processes. OPN expression is increased in multiple cancers, and OPN within tumour tissue has been shown to promote key stages of cancer development. OPN levels are also elevated in the circulation of cancer patients, which in some cases has been correlated with enhanced metastatic propensity and poor prognosis. However, the precise impact of circulating OPN (cOPN) on tumour growth and progression remains insufficiently understood. To examine the role of cOPN, we used a melanoma model, in which we stably increased the levels of cOPN through adeno-associated virus-mediated transduction. We found that increased cOPN promoted the growth of primary tumours, but did not significantly alter the spontaneous metastasis of melanoma cells to the lymph nodes or lungs, despite an increase in the expression of multiple factors linked to tumour progression. To assess whether cOPN has a role at later stages of metastasis formation, we employed an experimental metastasis model, but again could not detect any increase in pulmonary metastasis in animals with elevated levels of cOPN. These results demonstrate that increased levels of OPN in the circulation play distinct roles during different stages of melanoma progression.

Ketogenic diet does not promote triple-negative and luminal mammary tumor growth and metastasis in experimental mice.

Ketogenic diets (KDs) can improve the well-being and quality of life of breast cancer patients. However, data on the effects of KDs on mammary tumors are inconclusive, and the influence of KDs on metastasis in general remains to be investigated. We therefore assessed the impact of a KD on growth and metastasis of triple negative murine 4T1 mammary tumors, and on the progression of luminal breast tumors in an autochthonous MMTV-PyMT mouse model. We found that KD did not influence the metastasis of 4T1 and MMTV-PyMT mammary tumors, but impaired 4T1 tumor cell proliferation in vivo, and also temporarily reduced 4T1 primary tumor growth. Notably, the ketogenic ratio (the mass of dietary fat in relation to the mass of dietary carbohydrates and protein) that is needed to induce robust ketosis was twice as high in mice as compared to humans. Surprisingly, only female but not male mice responded to KD with a sustained increase in blood ß-hydroxybutyrate levels. Together, our data show that ketosis does not foster primary tumor growth and metastasis, suggesting that KDs can be safely applied in the context of luminal breast cancer, and may even be advantageous for patients with triple negative tumors. Furthermore, our data indicate that when performing experiments with KDs in mice, the ketogenic ratio needed to induce ketosis must be verified, and the sex of the mice should also be taken into account.

The crosstalk between glomerular endothelial cells and podocytes controls their responses to metabolic stimuli in diabetic nephropathy.

In diabetic nephropathy (DN), glomerular endothelial cells (GECs) and podocytes undergo pathological alterations, which are influenced by metabolic changes characteristic of diabetes, including hyperglycaemia (HG) and elevated methylglyoxal (MGO) levels. However, it remains insufficiently understood what effects these metabolic factors have on GEC and podocytes and to what extent the interactions between the two cell types can modulate these effects. To address these questions, we established a co-culture system in which GECs and podocytes were grown together in close proximity, and assessed transcriptional changes in each cell type after exposure to HG and MGO. We found that HG and MGO had distinct effects on gene expression and that the effect of each treatment was markedly different between GECs and podocytes. HG treatment led to upregulation of "immediate early response" genes, particularly those of the EGR family, as well as genes involved in inflammatory responses (in GECs) or DNA replication/cell cycle (in podocytes). Interestingly, both HG and MGO led to downregulation of genes related to extracellular matrix organisation in podocytes. Crucially, the transcriptional responses of GECs and podocytes were dependent on their interaction with each other, as many of the prominently regulated genes in co-culture of the two cell types were not significantly changed when monocultures of the cells were exposed to the same stimuli. Finally, the changes in the expression of selected genes were validated in BTBR ob/ob mice, an established model of DN. This work highlights the molecular alterations in GECs and podocytes in response to the key diabetic metabolic triggers HG and MGO, as well as the central role of GEC-podocyte crosstalk in governing these responses.

Fat Grafts Show Higher Hypoxia, Angiogenesis, Adipocyte Proliferation, and Macrophage Infiltration than Flaps in a Pilot Mouse Study.

BACKGROUND: Over 137,000 breast reconstructions are performed annually by American Society of Plastic Surgeons (ASPS) members. Vascularized flaps and avascular lipofilling each account for over 33,000 autologous reconstructions. Although clinical and experimental observations suggest biologic differences with diverging effects on locoregional tumor control, comparative animal models are lacking. The authors standardized existing techniques in immunocompetent mice, laying the foundation for in vivo models of autologous breast reconstruction combinable with orthotopic tumor implantations. METHODS: Twenty-five groin flaps and 39 fat grafts were transferred in female BALB/c-mice. Adipocytes were tracked via Hoechst-Calcein-DiI staining ( n = 2 per group), and postoperative volume retentions were compared via magnetic resonance imaging ( n = 3 per group) on days 1, 11, 21, and 31. Proliferation indices, microvessel densities, tissue hypoxia, and macrophage infiltrates were compared via Ki67, CD31, pimonidazole, and hematoxylin-eosin staining on days 5, 10, 15, 20, and 30 ( n = 4 per group). RESULTS: Viable adipocytes were present in both groups. Graft volumes plateaued at 42.7 ± 1.2% versus 81.8 ± 4.0% of flaps ( P < 0.001). Initially, grafts contained more hypoxic cells (day 5: 15.192 ± 1.249 versus 1.157 ± 192; P < 0.001), followed by higher proliferation (day 15: 25.2 ± 1.0% versus 0.0 ± 0.0%; P < 0.001), higher microvessel numbers (day 30: 307.0 ± 13.2 versus 178.0 ± 10.6; P < 0.001), and more pronounced macrophage infiltrates (graded 3 versus 2; P < 0.01). CONCLUSION: This comparative murine pilot study of vascularized flaps versus avascular lipofilling suggests differences in volume retention, proliferation, angiogenesis, hypoxia, and inflammation. CLINICAL RELEVANCE STATEMENT: The biological differences of fat grafting versus flap transfer are not fully understood because no single comparative experimental model has been established to date. The authors present the first comparative small animal model of both techniques, which will allow the gaining of deeper insights into their biological effects.

Molecular recognition of the Tes LIM2-3 domains by the actin-related protein Arp7A.

Actin-related proteins (Arps) are a highly conserved family of proteins that have extensive sequence and structural similarity to actin. All characterized Arps are components of large multimeric complexes associated with chromatin or the cytoskeleton. In addition, the human genome encodes five conserved but largely uncharacterized "orphan" Arps, which appear to be mostly testis-specific. Here we show that Arp7A, which has 43% sequence identity with ß-actin, forms a complex with the cytoskeletal proteins Tes and Mena in the subacrosomal layer of round spermatids. The N-terminal 65-residue extension to the actin-like fold of Arp7A interacts directly with Tes. The crystal structure of the 1-65(Arp7A)·LIM2-3(Tes)·EVH1(Mena) complex reveals that residues 28-49 of Arp7A contact the LIM2-3 domains of Tes. Two alanine residues from Arp7A that occupy equivalent apolar pockets in both LIM domains as well as an intervening GPAK linker that binds the LIM2-3 junction are critical for the Arp7A-Tes interaction. Equivalent occupied apolar pockets are also seen in the tandem LIM domain structures of LMO4 and Lhx3 bound to unrelated ligands. Our results indicate that apolar pocket interactions are a common feature of tandem LIM domain interactions, but ligand specificity is principally determined by the linker sequence.

Loss of ASAP1 in the MMTV-PyMT model of luminal breast cancer activates AKT, accelerates tumorigenesis, and promotes metastasis.

ASAP1 is a multi-domain adaptor protein that regulates cytoskeletal dynamics, receptor recycling and intracellular vesicle trafficking. Its expression is associated with poor prognosis in a variety of cancers, and can promote cell migration, invasion and metastasis. Although amplification and expression of ASAP1 has been associated with poor survival in breast cancer, we found that in the autochthonous MMTV-PyMT model of luminal breast cancer, ablation of ASAP1 resulted in an earlier onset of tumor initiation and increased metastasis. This was due to tumor cell-intrinsic effects of ASAP1 deletion, as ASAP1 deficiency in tumor, but not in stromal cells was sufficient to replicate the enhanced tumorigenicity and metastasis observed in the ASAP1-null MMTV-PyMT mice. Loss of ASAP1 in MMTV-PyMT mice had no effect on proliferation, apoptosis, angiogenesis or immune cell infiltration, but enhanced mammary gland hyperplasia and tumor cell invasion, indicating that ASAP1 can accelerate tumor initiation and promote dissemination. Mechanistically, these effects were associated with a potent activation of AKT. Importantly, lower ASAP1 levels correlated with poor prognosis and enhanced AKT activation in human ER+/luminal breast tumors, validating our findings in the MMTV-PyMT mouse model for this subtype of breast cancer. Taken together, our findings reveal that ASAP1 can have distinct functions in different tumor types and demonstrate a tumor suppressive activity for ASAP1 in luminal breast cancer.

Sulfated hyaluronic acid inhibits the hyaluronidase CEMIP and regulates the HA metabolism, proliferation and differentiation of fibroblasts.

Hyaluronan (HA) is an extracellular matrix component that regulates a variety of physiological and pathological processes. The function of HA depends both on its overall amount and on its size, properties that are controlled by HA synthesizing and degrading enzymes. The lack of inhibitors that can specifically block individual HA degrading enzymes has hampered attempts to understand the contribution of individual hyaluronidases to different physiological and pathological processes. CEMIP is a recently discovered hyaluronidase that cleaves HA through mechanisms and under conditions that are distinct from those of other hyaluronidases such as HYAL1 or HYAL2. The role of its hyaluronidase activity in physiology and disease is poorly understood. Here, we characterized a series of sulfated HA derivatives (sHA) with different sizes and degrees of sulfation for their ability to inhibit specific hyaluronidases. We found that highly sulfated sHA derivatives potently inhibited CEMIP hyaluronidase activity. One of these compounds, designated here as sHA3.7, was characterized further and shown to inhibit CEMIP with considerable selectivity over other hyaluronidases. Inhibition of CEMIP with sHA3.7 in fibroblasts, which are the main producers of HA in the interstitial matrix, increased the cellular levels of total and high molecular weight HA, while decreasing the fraction of low molecular weight HA fragments. Genetic deletion of CEMIP in mouse embryonic fibroblasts (MEFs) produced analogous results and confirmed that the effects of sHA3.7 on HA levels were mediated by CEMIP inhibition. Importantly, both CEMIP deletion and its inhibition by sHA3.7 suppressed fibroblast proliferation, while promoting differentiation into myofibroblasts, as reflected in a lack of CEMIP in myofibroblasts within skin wounds in experimental mice. By contrast, adipogenic and osteogenic differentiation were attenuated upon CEMIP loss or inhibition. Our results demonstrate the importance of CEMIP for the HA metabolism, proliferation and differentiation of fibroblasts, and suggest that inhibition of CEMIP with sulfated HA derivatives such as sHA3.7 has potential utility in pathological conditions that are dependent on CEMIP function.

Rac1 regulates neuronal polarization through the WAVE complex.

Neuronal migration and axon growth, key events during neuronal development, require distinct changes in the cytoskeleton. Although many molecular regulators of polarity have been identified and characterized, relatively little is known about their physiological role in this process. To study the physiological function of Rac1 in neuronal development, we have generated a conditional knock-out mouse, in which Rac1 is ablated in the whole brain. Rac1-deficient cerebellar granule neurons, which do not express other Rac isoforms, showed impaired neuronal migration and axon formation both in vivo and in vitro. In addition, Rac1 ablation disrupts lamellipodia formation in growth cones. The analysis of Rac1 effectors revealed the absence of the Wiskott-Aldrich syndrome protein (WASP) family verprolin-homologous protein (WAVE) complex from the plasma membrane of knock-out growth cones. Loss of WAVE function inhibited axon growth, whereas overexpression of a membrane-tethered WAVE mutant partially rescued axon growth in Rac1-knock-out neurons. In addition, pharmacological inhibition of the WAVE complex effector Arp2/3 also reduced axon growth. We propose that Rac1 recruits the WAVE complex to the plasma membrane to enable actin remodeling necessary for axon growth.

Luminal particles within cellular microtubules.

The regulation of microtubule dynamics is attributed to microtubule-associated proteins that bind to the microtubule outer surface, but little is known about cellular components that may associate with the internal side of microtubules. We used cryoelectron tomography to investigate in a quantitative manner the three dimensional structure of microtubules in intact mammalian cells. We show that the lumen of microtubules in this native state is filled with discrete, globular particles with a diameter of 7 nm and spacings between 8 and 20 nm in neuronal cells. Cross-sectional views of microtubules confirm the presence of luminal material in vitreous sections of brain tissue. Most of the luminal particles had connections to the microtubule wall, as revealed in tomograms. A higher accumulation of particles was seen near the retracting plus ends of microtubules. The luminal particles were abundant in neurons, but were also observed in other cells, such as astrocytes and stem cells.

A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha.

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

Quantitative Detection of Disseminated Melanoma Cells by Trp-1 Transcript Analysis Reveals Stochastic Distribution of Pulmonary Metastases.

A better understanding of the process of melanoma metastasis is required to underpin the development of novel therapies that will improve patient outcomes. The use of appropriate animal models is indispensable for investigating the mechanisms of melanoma metastasis. However, reliable and practicable quantification of metastases in experimental mice remains a challenge, particularly if the metastatic burden is low. Here, we describe a qRT-PCR-based protocol that employs the melanocytic marker Trp-1 for the sensitive quantification of melanoma metastases in the murine lung. Using this protocol, we were able to detect the presence of as few as 100 disseminated melanoma cells in lung tissue. This allowed us to quantify metastatic burden in a spontaneous syngeneic B16-F10 metastasis model, even in the absence of visible metastases, as well as in the autochthonous Tg(Grm1)/Cyld-/- melanoma model. Importantly, we also observed an uneven distribution of disseminated melanoma cells amongst the five lobes of the murine lung, which varied considerably from animal to animal. Together, our findings demonstrate that the qRT-PCR-based detection of Trp-1 allows the quantification of low pulmonary metastatic burden in both transplantable and autochthonous murine melanoma models, and show that the analysis of lung metastasis in such models needs to take into account the stochastic distribution of metastatic lesions amongst the lung lobes.

IER2-induced senescence drives melanoma invasion through osteopontin.

Expression of the immediate-early response gene IER2 has been associated with the progression of several types of cancer, but its functional role is poorly understood. We found that increased IER2 expression in human melanoma is associated with shorter overall survival, and subsequently investigated the mechanisms through which IER2 exerts this effect. In experimental melanoma models, sustained expression of IER2 induced senescence in a subset of melanoma cells in a p53/MAPK/AKT-dependent manner. The senescent cells produced a characteristic secretome that included high levels of the extracellular phosphoglycoprotein osteopontin. Nuclear localization of the IER2 protein was critical for both the induction of senescence and osteopontin secretion. Osteopontin secreted by IER2-expressing senescent cells strongly stimulated the migration and invasion of non-senescent melanoma cells. Consistently, we observed coordinate expression of IER2, p53/p21, and osteopontin in primary human melanomas and metastases, highlighting the pathophysiological relevance of IER2-mediated senescence in melanoma progression. Together, our study reveals that sustained IER2 expression drives melanoma invasion and progression through stimulating osteopontin secretion via the stochastic induction of senescence.