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PDF file 110K, Figure legends for Supplementary Data

PDF file 103K, Decreasing ROR1 increases ROR2 and decreases proliferation in vivo

PDF file 197K, Table of patient characteristics, and Wnt5A staining in paired samples pre and post-therapy

PDF file 283K, Table of patient characteristics, and Wnt5A staining, pre-therapy

An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance.Significance: These data show for the first time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib. Cancer Discov; 3(12); 1378–93. ©2013 AACR.This article is highlighted in the In This Issue feature, p. 1317

Supplementary Tables

Supplementary Figure 4

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma.Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals.Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor–sensitive and BRAF inhibitor–resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors.Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181–90. ©2017 AACR.

Legends to supplementary figures

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Supplementary Figure 3

Objectives Moderately active RA is associated with poor patient outcomes. Despite this, some health systems have restricted access to advanced therapies to those with severe RA. There is also limited evidence of the efficacy of advanced therapies in the moderately active RA population. This post-hoc analysis from four phase 3 trials explored the efficacy of upadacitinib (UPA) for moderately active RA. Methods Patients included in this analysis received UPA 15 mg once daily [monotherapy after switching from MTX or in combination with stable background conventional synthetic DMARDs (csDMARDs)] or placebo. Clinical, functional and radiographic outcomes were analysed separately for patients with moderate disease activity {28-joint count DAS using CRP [DAS28(CRP)] of >3.2 and ≤5.1} and severe disease activity [DAS28(CRP) >5.1]. Results Patients with moderate disease activity who received UPA 15 mg (combination or monotherapy) after an inadequate response to biologic DMARDs and/or csDMARDs were significantly more likely to achieve a 20% improvement in the ACR response criteria, low disease activity status [DAS28(CRP) ≤ 3.2] or clinical remission [DAS28(CRP) Conclusion This analysis provides support for the use of UPA for the treatment of patients with moderate RA. Trial registration ClinicalTrials.gov: SELECT-NEXT: NCT02675426; SELECT-COMPARE: NCT02629159; SELECT-MONOTHERAPY: NCT02706951; SELECT-BEYOND: NCT02706847.

Purpose: Aging is a poor prognostic factor for melanoma. We have shown that melanoma cells in an aged microenvironment are more resistant to targeted therapy than identical cells in a young microenvironment. This is dependent on age-related secreted factors. Klotho is an age-related protein whose serum levels decrease dramatically by age 40. Most studies on klotho in cancer have focused on the expression of klotho in the tumor cell. We have shown that exogenous klotho inhibits internalization and signaling of Wnt5A, which drives melanoma metastasis and resistance to targeted therapy. We investigate here whether increasing klotho in the aged microenvironment could be an effective strategy for the treatment of melanoma. Experimental Design: PPARγ increases klotho levels and is increased by glitazones. Using rosiglitazone, we queried the effects of rosiglitazone on Klotho/Wnt5A cross-talk, in vitro and in vivo, and the implications of that for targeted therapy in young versus aged animals. Results: We show that rosiglitazone increases klotho and decreases Wnt5A in tumor cells, reducing the burden of both BRAF inhibitor–sensitive and BRAF inhibitor–resistant tumors in aged, but not young mice. However, when used in combination with PLX4720, tumor burden was reduced in both young and aged mice, even in resistant tumors. Conclusions: Using glitazones as adjuvant therapy for melanoma may provide a new treatment strategy for older melanoma patients who have developed resistance to vemurafenib. As klotho has been shown to play a role in other cancers too, our results may have wide relevance for multiple tumor types. Clin Cancer Res; 23(12); 3181–90. ©2017 AACR.

We have previously shown that Wnt5A drives invasion in melanoma. We have also shown that Wnt5A promotes resistance to therapy designed to target the BRAF(V600E) mutation in melanoma. Here, we show that melanomas characterized by high levels of Wnt5A respond to therapeutic stress by increasing p21 and expressing classical markers of senescence, including positivity for senescence-associated β-galactosidase (SA-β-gal), senescence-associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. We find that despite this, these cells retain their ability to migrate and invade. Further, despite the expression of classic markers of senescence such as SA-β-gal and SAHF, these Wnt5A-high cells are able to colonize the lungs in in vivo tail vein colony-forming assays. This clearly underscores the fact that these markers do not indicate true senescence in these cells, but instead an adaptive stress response that allows the cells to evade therapy and invade. Notably, silencing Wnt5A reduces expression of these markers and decreases invasiveness. The combined data point to Wnt5A as a master regulator of an adaptive stress response in melanoma, which may contribute to therapy resistance.

Cancer is a disease of ageing. Clinically, aged cancer patients tend to have a poorer prognosis than young. This may be due to accumulated cellular damage, decreases in adaptive immunity, and chronic inflammation. However, the effects of the aged microenvironment on tumour progression have been largely unexplored. Since dermal fibroblasts can have profound impacts on melanoma progression, we examined whether age-related changes in dermal fibroblasts could drive melanoma metastasis and response to targeted therapy. Here we find that aged fibroblasts secrete a Wnt antagonist, sFRP2, which activates a multi-step signalling cascade in melanoma cells that results in a decrease in β-catenin and microphthalmia-associated transcription factor (MITF), and ultimately the loss of a key redox effector, APE1. Loss of APE1 attenuates the response of melanoma cells to DNA damage induced by reactive oxygen species, rendering the cells more resistant to targeted therapy (vemurafenib). Age-related increases in sFRP2 also augment both angiogenesis and metastasis of melanoma cells. These data provide an integrated view of how fibroblasts in the aged microenvironment contribute to tumour progression, offering new possibilities for the design of therapy for the elderly.

The incidence of malignant melanoma, a cancer of the melanocyte cell lineage, has nearly doubled in the past 20 years. Wnt5A, a key driver of melanoma invasiveness, induces Ca2+ signals. To understand how store-operated calcium entry (SOCE) contributes to Wnt5A-induced malignancy in melanoma models, we examined the expression and function of STIM1 and Orai1 in patient-derived malignant melanoma cells, previously characterized as either highly invasive (metastatic) or noninvasive. Using both fluorescence microscopy and electrophysiological approaches, we show that SOCE is greatly diminished in invasive melanoma compared to its level in noninvasive cell types. However, no loss of expression of any members of the STIM and Orai families was observed in invasive melanoma cells. Moreover, overexpressed wild-type STIM1 and Orai1 failed to restore SOCE in invasive melanoma cells, and we observed no defects in their localization before or after store depletion in any of the invasive cell lines. Importantly, however, we determined that SOCE was restored by inhibition of protein kinase C, a known downstream target of Wnt5A. Furthermore, coexpression of STIM1 with an Orai1 mutant insensitive to protein kinase C-mediated phosphorylation fully restored SOCE in invasive melanoma. These findings reveal a level of control for STIM/Orai function in invasive melanoma not previously reported.

Skeletal remodeling is an ongoing process requiring the coordinated action of different cell types to maintain homeostatic control of bone synthesis and degradation. Mutations in p62/SQSTM1 are associated with sporadic and 5q35-linked Paget Disease of Bone (PDB), characterized by focal increased bone turnover. These mutations cluster in the ubiquitin associated (UBA) domain and are thought to lead to enhancement of NFκB pathway activation involved in osteoclastogenesis and hyper-responsiveness to receptor activator of nuclear factorκB ligand (RANKL). The structurally similar selective autophagic receptor, Nbr1, binds to LC3 and p62 and is sequestered into autophagosomes, whereas it accumulates in autophagic-deficient tissues. We have shown that truncation of Nbr1 in a murine model, where it can still interact with p62 but not LC3, leads to increased osteoblast differentiation and activity in vivo. This results in an age-dependent increase in bone mass and bone mineral density. This is a molecular consequence of loss of autophagy receptor function via deletion of its C-terminal UBA domain, and/or modulation of the p38 MAPK cellular signaling pathway.

An emerging concept in melanoma biology is that of dynamic, adaptive phenotype switching, where cells switch from a highly proliferative, poorly invasive phenotype to a highly invasive, less proliferative one. This switch may hold significant implications not just for metastasis, but also for therapy resistance. We demonstrate that phenotype switching and subsequent resistance can be guided by changes in expression of receptors involved in the noncanonical Wnt5A signaling pathway, ROR1 and ROR2. ROR1 and ROR2 are inversely expressed in melanomas and negatively regulate each other. Furthermore, hypoxia initiates a shift of ROR1-positive melanomas to a more invasive, ROR2-positive phenotype. Notably, this receptor switch induces a 10-fold decrease in sensitivity to BRAF inhibitors. In patients with melanoma treated with the BRAF inhibitor vemurafenib, Wnt5A expression correlates with clinical response and therapy resistance. These data highlight the fact that mechanisms that guide metastatic progression may be linked to those that mediate therapy resistance. Significance: These data show for the first time that a single signaling pathway, the Wnt signaling pathway, can effectively guide the phenotypic plasticity of tumor cells, when primed to do so by a hypoxic microenvironment. Importantly, this increased Wnt5A signaling can give rise to a subpopulation of highly invasive cells that are intrinsically less sensitive to novel therapies for melanoma, and targeting the Wnt5A/ROR2 axis could improve the efficacy and duration of response for patients with melanoma on vemurafenib. Cancer Discov; 3(12); 1378–93. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 1317

Nature 532, 250–254 (2016); doi:10.1038/nature17392 In Fig. 5a of this Letter, the labels PBS and PLX4720 were inadvertently reversed. The corrected Fig. 5a is shown in Fig. 1 of this Corrigendum.

The neighbor of Brca1 gene (Nbr1) functions as an autophagy receptor involved in targeting ubiquitinated proteins for degradation. It also has a dual role as a scaffold protein to regulate growth-factor receptor and downstream signaling pathways. We show that genetic truncation of murine Nbr1 leads to an age-dependent increase in bone mass and bone mineral density through increased osteoblast differentiation and activity. At 6 mo of age, despite normal body size, homozygous mutant animals (Nbr1 tr/tr ) have ~50% more bone than littermate controls. Truncated Nbr1 (trNbr1) co-localizes with p62, a structurally similar interacting scaffold protein, and the autophagosome marker LC3 in osteoblasts, but unlike the full-length protein, trNbr1 fails to complex with activated p38 MAPK. Nbr1 tr/tr osteoblasts and osteoclasts show increased activation of p38 MAPK, and significantly, pharmacological inhibition of the p38 MAPK pathway in vitro abrogates the increased osteoblast differentiation of Nbr1 tr/tr cells. Nbr1 truncation also leads to increased p62 protein expression. We show a role for Nbr1 in bone remodeling, where loss of function leads to perturbation of p62 levels and hyperactivation of p38 MAPK that favors osteoblastogenesis.

Background: Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. The expression level of klotho, an emerging tumor suppressor, declines with the progression of age and is associated with several pathologies related to human aging. A previous study from our group has demonstrated that loss of klotho in melanoma cells is associated with enhanced melanoma cell motility by decreasing the ability of melanoma cells to internalize Wnt5A, a non-canonical Wnt protein, known to be one of the drivers of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling and ultimately results in age-related melanoma aggression. Methods: In order to study the effects of aged microenvironment on melanoma cells, we have obtained and cultured normal skin fibroblasts from aged (55-65 years old) and young donors (25-35 years old). The expression levels of klotho and Wnt5A in these fibroblasts were analyzed by immunoblot, immunofluorescence and real time-PCR assays. We examined melanoma cell/ fibroblast co-cultures and artificial skin reconstructs made with these fibroblasts to understand the age-related effects of tumor microenvironment on the disease progression. We also used immunohistochemistry technique to examine the expression levels of Klotho and Wnt5A in these artificial skin reconstructs and in the paraffin-embedded skin sections obtained from young and old melanoma patients. Results: Our western blot and real-time PCR results of the young vs. old fibroblasts show that klotho expression level is lost with progression of age. Analysis of artificial skin reconstructs made using skin fibroblasts isolated from aged donors demonstrate induced invasion of melanoma cells to a much greater extent than those cultured from young donors. We also analyzed these skin reconstructs for the expression levels of Klotho and Wnt5A, using immunohistochemistry technique and the results confirm that Klotho is decreased in aged fibroblasts. Furthermore, we also found that the loss of Klotho in the aged microenvironment translated to a loss of Klotho in melanoma cells. Our western blot results also suggest that loss of Klotho in the aging microenvironment enhances Wnt5A internalization and signaling and this promotes tumor metastasis. Our immunohistochemistry results using patient samples further extend our in vitro findings and support our hypothesis that Klotho plays pivotal role in age-related melanoma progression. Significance: These results for the first time demonstrate a correlation between age-related melanoma progression and the loss of klotho level. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Marie Webster, Xiaowei Xu, Ashani T. Weeraratna. Role of Klotho in age-related melanoma progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1556. doi:10.1158/1538-7445.AM2015-1556

Background: Klotho, an emerging tumor suppressor, was originally identified as an anti-aging gene. The klotho expression level declines with the progression of age and is associated with several pathologies related to human aging. Mortality rates due to melanoma are continuously increasing. Specifically, in older patients, melanoma is very aggressive and has a very poor prognosis, compared to the young. Therefore, there is an urgent need to understand the underlying molecular mechanisms behind this age-related aggression. A previous study from our group has demonstrated that loss of klotho in melanoma cells is associated with enhanced melanoma cell motility by decreasing the ability of melanoma cells to internalize Wnt5A, a non-canonical Wnt protein, known to be a driver of melanoma metastasis. Therefore, we hypothesize that the loss of Klotho in aging microenvironment contributes to enhanced Wnt5A signaling, ultimately resulting in age-related melanoma aggression. Methods: In order to study the effects of aged microenvironment on melanoma cells, we have obtained and cultured normal skin fibroblasts from aged (55-65 years old) and young donors (25-35 years old) from the Baltimore Longitudinal Study of Aging (BLSA). The expression levels of klotho and Wnt5A in these fibroblasts were analyzed by immunoblot, immunofluorescence and real time-PCR assays. We examined melanoma cell/ fibroblast co-cultures and artificial skin reconstructs made with these fibroblasts to understand the age-related effects of tumor microenvironment on the disease progression. We also used immunohistochemistry technique to examine the expression levels of Klotho and Wnt5A in these artificial skin reconstructs and in the paraffin-embedded skin sections obtained from young and old melanoma patients. Results: Our western blot and real-time PCR results of the young vs. old fibroblasts show that klotho expression level is lost and Wnt5A expression is gained with progression of age. Analysis of artificial skin reconstructs made using skin fibroblasts isolated from aged donors demonstrate induced invasion of melanoma cells to a much greater extent than those cultured from young donors. We also analyzed these skin reconstructs for the expression levels of Klotho and Wnt5A, using immunohistochemistry technique and our results confirm that Klotho is decreased in aged fibroblasts, furthermore, we also found that the loss of Klotho in the aged microenvironment also gets translated to a loss of Klotho in melanoma cells, and a gain of Wnt5A. Our immunohistochemistry results with the patient samples further extend our in vitro findings and support our hypothesis that Klotho-Wnt5A cross talk plays pivotal role in age-related melanoma progression. Significance: These results for the first time demonstrate a correlation between age-related melanoma progression and the loss of klotho and gain in Wnt5A expression levels in the aging tumor microenvironment. In addition, these findings provide a molecular explanation of age-related melanoma aggression. Citation Format: Reeti Behera, Katie Marchbank, Amanpreet Kaur, Vanessa Dang, Marie Webster, Michael O'Connell, Xiaowei Xu, Ashani Weeraratna. Crosstalk between klotho and wnt5A drives age-related melanoma progression. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A11.

Senescence is a cytostatic cell fate that can be induced by oncogene activation, replicative stress or therapy-induced stress. Therapy-induced senescence (TIS) of tumor cells can be initiated by anti-cancer compounds or radiation. TIS is generally considered to be irreversible, and as such this process is believed to be a desirable therapeutic outcome. In this study, we show that melanomas characterized by high levels of the signaling molecule Wnt5A respond to irradiation by undergoing growth arrest, and by expressing classical markers of senescence, including positivity for senescence-associated beta-galactosidase (SA-β-gal), senescence associated heterochromatic foci (SAHF), H3K9Me chromatin marks, and PML bodies. Even though these cells are positive for classical markers of senescence, we find that they retain their ability to migrate and invade. Further, these Wnt5A-high cells are able to proliferate following metastasis and form new tumors in the lung, while expressing increased markers of senescence. Silencing Wnt5A reduces expression of these senescence markers and decreases invasiveness. Finally, Wnt5A-high melanoma cells treated with PLX4720, a BRAFV600E targeted therapy, also increase markers of a TIS response, while remaining invasive. The combined data challenge the paradigm that TIS is a desirable therapeutic endpoint. They also point to Wnt5A as a master regulator of this senescence-like stress-induced phenotype in melanoma. Citation Format: Marie R. Webster, Mai Xu, Kathryn Kinzler, Amanpreet Kaur, Jessica Appleton, Michael P. O'Connell, Katie Marchbank, Alexander Valiga, Vanessa Dang, Michela Perego, Gao Zhang, Ana Slipicevic, Frederick Keeney, Elin Lehrmann, William Wood, III, Kevin Becker, Andrew V. Kossenkov, Meenhard Herlyn, Maureen Murphy, Ashani T. Weeraratna. Wnt5A-expressing melanoma cells show classical markers of senescence following radiation and therapeutic stress, but retain the ability to metastasize and proliferate at distant sites. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr B27.

The incidence of melanoma rises dramatically after the age of 55. Due to an increase in aging population, it is important to study the change in molecular mechanisms due to aging that would allow development of therapies that are tailored to the age of the patients. The role of the tumor microenvironment in modulating cancer characteristics is widely recognized and it also provides targets for therapeutic intervention. Due to this, we hypothesized that changes in tumor microenvironment due to aging could affect the progression of the melanoma. We obtained skin fibroblasts from healthy donors aged 25-35, as well as skin fibroblasts from healthy donors aged 55-65. We cultured these fibroblasts and used conditioned media from them to affect the invasion of melanoma cells in 3D spheroid invasion assays, where aged fibroblasts promoted invasion of melanoma cells into collagen. We also built artificial skin (reconstructs) using young and aged fibroblasts and demonstrated that skin built with aged fibroblasts promoted melanoma cell invasion. Finally using a transgenic mouse model of melanoma (Yumm1.7, BRAFV600E/PTEN-/-) we observed that the injection of melanoma cells into the tail vein of aged mice (52 weeks) formed metastastic colonies much more rapidly than those injected into the tail vein of young mice (8 weeks). To study the factors involved in the aging microenvironment, we performed a proteomics study of the secretome from young and aged fibroblasts. From this study, we observed that aged fibroblasts secreted inhibitors of canonical Wnt signaling, as well as increased deposition of extracellular matrix components in the aging microenvironment. Since inhibition of canonical Wnt signaling has been linked to decreased sensitivity towards BRAF inhibitors in melanoma, we injected Yumm 1.7 cells subcutaneously into aged and young mice. These mice were then treated with BRAF inhibitors. We observed an increased resistance in response to therapy in the aged mice. We also prepared skin reconstruct from fibroblasts with knockdown of the proteins that we identified from secretome and treated them with PLX4720. These results indicated the role of these extracellular matrix proteins in melanoma. We are exploring the mechanisms of how these extracellular matrix proteins affect the sensitivity towards chemotherapeutics. We concluded that aging could alter tumor microenvironment thus resulting in increased metastasis and therapy resistance. It is important that the studies in cancer therapies take into account the age of the patient to achieve better response in patients. Citation Format: Amanpreet Kaur, Katie Marchbank, Vanessa Dang, Michael O'Connell, Marie Webster, Jessica Appleton, Phil Cheng, Alexander Valiga, Rachel Morissette, Nazli McDonnell, Luigi Ferrucci, Andrew Kossenkov, Katrina Meeth, Marcus Bosenberg, Hsin-Yao Tang, Xiangfan Yin, William Wood, III, Elin Lehrmann, Kevin Becker, Keith Flaherty, Dennie Frederick, Jennifer Wargo, Katherine Aird, Rugang Zhang, Xiaowei Xu, Qin Liu, David Speicher, Ashani Weeraratna. Aging microenvironment modulates melanoma invasion and metastasis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A04.