Older melanoma patients have lower rates of sentinel lymph node (LN) metastases yet paradoxically have inferior survival. Patient age correlated with an inability to retain Technetium radiotracer during sentinel LN biopsy in over 1000 patients, and high technecium counts correlated to better survival. We hypothesized that loss of integrity in the lymphatic vasculature due to ECM degradation might play a role. We have implicated HAPLN1 in age-dependent ECM degradation in the dermis. Here we queried whether HAPLN1 could be altered in the lymphatic ECM. Lymphatic HAPLN1 expression was prognostic of long-term patient survival. Adding rHAPLN1 to aged fibroblast ECMs in vitro reduced endothelial permeability via modulation of VE-Cadherin junctions, whereas endothelial permeability was increased following HAPLN1-knockdown in young fibroblasts. In vivo, reconstitution of HAPLN1 in aged mice increased the number of LN metastases, but reduced visceral metastases. These data suggest that age-related changes in ECM can contribute to impaired lymphatics.

To date, no consistent oncogenic driver mutations have been identified in most adult soft tissue sarcomas; these tumors are thus generally insensitive to existing targeted therapies. Here we investigated alternate mechanisms underlying sarcomagenesis to identify potential therapeutic interventions. Undifferentiated pleomorphic sarcoma (UPS) is an aggressive tumor frequently found in skeletal muscle where deregulation of the Hippo pathway and aberrant stabilization of its transcriptional effector yes-associated protein 1 (YAP1) increases proliferation and tumorigenesis. However, the downstream mechanisms driving this deregulation are incompletely understood. Using autochthonous mouse models and whole genome analyses, we found that YAP1 was constitutively active in some sarcomas due to epigenetic silencing of its inhibitor angiomotin (AMOT). Epigenetic modulators vorinostat and JQ1 restored AMOT expression and wild-type Hippo pathway signaling, which induced a muscle differentiation program and inhibited sarcomagenesis. YAP1 promoted sarcomagenesis by inhibiting expression of ubiquitin-specific peptidase 31 (USP31), a newly identified upstream negative regulator of NFκB signaling. Combined treatment with epigenetic modulators effectively restored USP31 expression, resulting in decreased NFκB activity. Our findings highlight a key underlying molecular mechanism in UPS and demonstrate the potential impact of an epigenetic approach to sarcoma treatment. A new link between Hippo pathway signaling, NFκB, and epigenetic reprogramming is highlighted and has the potential for therapeutic intervention in soft tissue sarcomas. .

Dormant tumor cells escape the primary site, do not grow out into macroscopic tumors in the distal site, but maintain enough plasticity to reactivate and form overt metastatic lesions, sometimes taking several decades. Despite its importance in metastasis and residual disease, few studies have been able to successfully model or characterize dormancy within melanoma. Here, we show that age-related changes in the lung microenvironment facilitate a permissive niche for e cient outgrowth of disseminated dormant tumor cells, in contrast to the aged skin, where age-related changes suppress melanoma growth but drive dissemination. A model of melanoma progression that addresses these microenvironmental complexities is the phenotype switching model, which argues that melanoma cells switch between a proliferative cell state and a slower-cycling, invasive state 1-3 . Dermal broblasts are key orchestrators of promoting phenotype switching in melanoma via changes in the secretion of soluble factors during aging [4][5][6][7][8] . Speci cally, we have identi ed Wnt5A as a master regulator of activating metastatic dormancy, which enables e cient seeding and survival of melanoma cells in metastatic niches. Age-induced reprogramming of lung broblasts increases their secretion of the soluble Wnt antagonist sFRP1, which inhibits Wnt5A, enabling e cient metastatic outgrowth. Further, we have identi ed the tyrosine kinase receptors AXL and MER as promoting a dormancy-to-reactivation axis respectively. Overall, we nd that age-induced changes in distal metastatic microenvironments promotes e cient reactivation of dormant melanoma cells in the lung. MainWe have previously established that melanoma cells implanted in aged mouse skin metastasize to the lung at greater rates than in younger animals 4 . Whether this is due to increased dissemination from the primary site, or because the aged microenvironment at metastatic sites promotes outgrowth remained unclear. To investigate this, we intradermally implanted Yumm1.7 (mCherry) melanoma cells into young (8 weeks) and aged (> 52 weeks) C57BL6 mice. The primary tumor in the skin grew faster in young mice (Fig. 1A). We examined distal lung metastases at weeks 1, 3 and 5 using immunohistochemical (IHC) analysis of mCherry positive cells in the lung. At week 1, we failed to detect melanoma cells. At week 3, we found that melanoma cells e ciently seed the lung in equal numbers in young and aged mice (Fig. 1B) as single cell colonies (Fig. 1C, top panels); however, at week 5, larger metastatic colonies formed in the aged lung (Fig. 1C, bottom right), while cells persisted as single cells in the young (Fig. 1C, bottom left). While the number of cells that seeded in the young vs. aged lung are similar, the rate at which cells seed the lung (no. of cells disseminating/mm 3 of tumor volume) was far lower in young mice. Thus, to determine whether the difference in lung outgrowth at week 5 was due to an overall increase in dissemination from aged primary tumors, we removed the primary tumors from aged m...