Abibatou Ndoye

sFRP2 in the aged microenvironment drives melanoma metastasis and 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.

Response to Programmed Cell Death-1 Blockade in a Murine Melanoma Syngeneic Model Requires Costimulation, CD4, and CD8 T Cells.

Although blockade of the PD-1 pathway has been successfully used to treat various cancers, how this modulates host–tumor interactions is not well understood. Additional mechanisms beyond licensing the final effector phase of killer T cells were identified. The programmed cell death protein 1 (PD-1) limits effector T-cell functions in peripheral tissues, and its inhibition leads to clinical benefit in different cancers. To better understand how PD-1 blockade therapy modulates the tumor–host interactions, we evaluated three syngeneic murine tumor models, the BRAFV600E-driven YUMM1.1 and YUMM2.1 melanomas, and the carcinogen-induced murine colon adenocarcinoma MC38. The YUMM cell lines were established from mice with melanocyte-specific BRAFV600E mutation and PTEN loss (BRAFV600E/PTEN−/−). Anti–PD-1 or anti–PD-L1 therapy engendered strong antitumor activity against MC38 and YUMM2.1, but not YUMM1.1. PD-L1 expression did not differ between the three models at baseline or upon interferon stimulation. Whereas mutational load was high in MC38, it was lower in both YUMM models. In YUMM2.1, the antitumor activity of PD-1 blockade had a critical requirement for both CD4 and CD8 T cells, as well as CD28 and CD80/86 costimulation, with an increase in CD11c+CD11b+MHC-IIhigh dendritic cells and tumor-associated macrophages in the tumors after PD-1 blockade. Compared with YUMM1.1, YUMM2.1 exhibited a more inflammatory profile by RNA sequencing analysis, with an increase in expression of chemokine-trafficking genes that are related to immune cell recruitment and T-cell priming. In conclusion, response to PD-1 blockade therapy in tumor models requires CD4 and CD8 T cells and costimulation that is mediated by dendritic cells and macrophages. Cancer Immunol Res; 4(10); 845–57. ©2016 AACR.

Autophagy- An emerging target for melanoma therapy

Melanoma accounts for only 5% of all cancers but is the leading cause of skin cancer death due to its high metastatic potential. Patients with metastatic melanoma have a 10-year survival rate of less than 10%. While the clinical landscape for melanoma is evolving rapidly, lack of response to therapies, as well as resistance to therapy remain critical obstacles for treatment of this disease. In recent years, a myriad of therapy resistance mechanisms have been unravelled, one of which is autophagy, the focus of this review. In advanced stages of malignancy, melanoma cells hijack the autophagy machinery in order to alleviate drug-induced and metabolic stress in the tumor microenvironment, thereby promoting resistance to multiple therapies, tumor cell survival, and progression. Autophagy is an essential cellular process that maintains cellular homeostasis through the recycling of intracellular constituents. Early studies on the role of autophagy in cancer generated controversy as to whether autophagy was pro- or anti-tumorigenic. Currently, there is a consensus that autophagy is tumor-suppressive in the early stages of cancer and tumor-promoting in established tumors. This review aims to highlight current understandings on the role of autophagy in melanoma malignancy, and specifically therapy resistance; as well as to evaluate recent strategies for therapeutic autophagy modulation.

Age Correlates with Response to Anti-PD1, Reflecting Age-Related Differences in Intratumoral Effector and Regulatory T-Cell Populations

Purpose: We have shown that the aged microenvironment increases melanoma metastasis, and decreases response to targeted therapy, and here we queried response to anti-PD1. Experimental Design: We analyzed the relationship between age, response to anti-PD1, and prior therapy in 538 patients. We used mouse models of melanoma, to analyze the intratumoral immune microenvironment in young versus aged mice and confirmed our findings in human melanoma biopsies. Results: Patients over the age of 60 responded more efficiently to anti-PD-1, and likelihood of response to anti-PD-1 increased with age, even when we controlled for prior MAPKi therapy. Placing genetically identical tumors in aged mice (52 weeks) significantly increased their response to anti-PD1 as compared with the same tumors in young mice (8 weeks). These data suggest that this increased response in aged patients occurs even in the absence of a more complex mutational landscape. Next, we found that young mice had a significantly higher population of regulatory T cells (Tregs), skewing the CD8+:Treg ratio. FOXP3 staining of human melanoma biopsies revealed similar increases in Tregs in young patients. Depletion of Tregs using anti-CD25 increased the response to anti-PD1 in young mice. Conclusions: While there are obvious limitations to our study, including our inability to conduct a meta-analysis due to a lack of available data, and our inability to control for mutational burden, there is a remarkable consistency in these data from over 500 patients across 8 different institutes worldwide. These results stress the importance of considering age as a factor for immunotherapy response. Clin Cancer Res; 24(21); 5347–56. ©2018 AACR. See related commentary by Pawelec, p. 5193

Inhibition of Age-Related Therapy Resistance in Melanoma by Rosiglitazone-Mediated Induction of Klotho

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.

Abstract 4913: Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation

Metastatic melanoma is highly aggressively and often therapy resistant. Signaling pathways which promote invasion of metastatic melanoma, also promote therapy resistance. Therapy resistant melanomas are characterized by high levels of the pro-invasive non-canonical Wnt molecule, Wnt5A. Previously, we described an adaptive stress response in highly invasive melanoma cells, which is characterized by a growth arrest and an increase in senescence markers, yet these cells retain the ability to invade and form colonies. These highly invasive cells do not undergo apoptosis following treatment with DNA damaging agents such as doxorubicin and are resistant to BRAFV600E targeted therapy. Recently, Lukin et al. have shown that p53 expression promotes survival of colorectal cancer cells via p21 expression and a reversible cell cycle arrest, allowing for repair of damaged DNA. Here, we show that highly invasive and therapy resistant melanoma cells express Wnt5A, p53 and p21, which regulate proliferation and slow cycling in invasive melanoma cells. The expression of p53 is promoted by Wnt5A, knock down of Wnt5A decreases p53 and p21 expression in these cells and decreases the number of cells arrested in G2/M following DNA damage. The cell cycle and apoptotic functions of p53 are highly regulated. iASPP, MDMX, and MDM2 have been shown to regulate the function of wild type p53 in melanoma cells. MDM2, an E4 ubiquitin ligase, regulates p53 by shuttling it out of the nucleus and targeting it for proteasomal degradation. We found that MDM2 expression increases in invasive melanoma cells following DNA damage, however, in these cells it is phosphorylated at serine 395. Phosphorylation of MDM2 at ser395 blocks its ability to export p53 from the nucleus, leading to increased p53 expression. Even with increased p53 expression, invasive melanoma cells undergo a cell cycle arrest following stress instead of apoptosis. The apoptotic function of p53 has been shown to be inhibited by nuclear iASPP, which is enriched in metastatic melanoma. We found that knocking down Wnt5A in invasive melanoma cells decreases the expression of iASPP. These data suggest that Wnt5A promotes MDM2 Ser395 phosphorylation and iASPP expression, blocking down regulation of p53 and its apoptotic function, while promoting cell cycle arrest and survival following stress. These data may reveal a mechanism by which highly invasive melanoma cells evade therapy to form therapy resistant sub-colonies at distant sites. Citation Format: Marie R. Webster, Amanpreet Kaur, Abibatou Ndoye, Curtis Kugel, Subhasree Basu, Alexander Valiga, Jessica Appleton, Ying-Jie Wang, Maureen Murphy, Ashani T. Weeraratna. Invasive melanoma cells commandeer p53 activity to promote the survival of a therapy resistant subpopulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4913. doi:10.1158/1538-7445.AM2017-4913

ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiologic relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. Cancer Res; 77(21); 5873-85. ©2017 AACR.

Corrigendum: sFRP2 in the aged microenvironment drives melanoma metastasis and therapy resistance

This corrects the article DOI: 10.1038/nature17392

Abstract 2906: Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis

Melanoma is the most aggressive type of skin cancer and the leading cause of death from skin disease. With increasing incidence of the disease, it is crucial to further investigate the cellular mechanisms and molecular pathways that lead to invasion, metastasis, and drug resistance. Recent studies have revealed that high autophagy correlates with melanoma tumor aggressiveness and poor survival in clinical samples, as it is a common mechanism of resistance to therapy. Autophagy inhibition leads to reduced levels in Wnt5A in a breast cancer model, suggesting a cross-talk of Wnt5A and autophagy in cancer. β catenin, a mediator of canonical Wnt signaling, has been shown to act as a negative regulator of both basal and induced autophagy in a colorectal cancer model. Our laboratory showed that high levels of Wnt5A correlate with increased invasion and metastasis in melanoma and that Wnt5A downregulates β catenin; therefore, we hypothesize that autophagy might drive invasion and metastasis in aggressive melanoma through the regulation of Wnt signaling. To study the role of autophagy in Wnt5A-mediated melanoma invasion, we inhibited autophagy in highly invasive melanoma cells using lentivirus-mediated shATG5 knockdown or hydroxychloroquine and evaluated the effects of autophagy inhibition on Wnt5A expression, β catenin, and invasion using 3D spheroid models. Analysis of autophagy flux confirms that highly invasive melanoma cells have high autophagy compared to non-invasive cell lines. The inhibition of autophagy by hydroxychloroquine or shATG5 both resulted in significant decrease in invasion. By western blot analysis, we also observed a decrease in Wnt5A in these cells. These results demonstrate that autophagy inhibition in highly invasive melanoma leads to decrease in invasion in a 3D model that mimics the tumor microenvironment of melanoma. Further dissection of the molecular mechanisms that are involved in this process will enable the identification of novel autophagy targets in aggressive melanoma. Citation Format: Abibatou Ndoye, Anna Budina, Marie Webster, Amanpreet Kaur, Reeti Behera, Maureen Murphy, Ashani T. Weeraratna. Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis. [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 2906. doi:10.1158/1538-7445.AM2015-2906

Abstract 5092: Midkine as a potential target for combating drug resistance and invasion in melanoma

Melanoma is an aggressive disease. Although about 13% of melanoma cases are diagnosed at metastatic stage, the 5-year survival rate for these patients is 16%. We have previously shown that metastatic melanoma cells express high levels of Wnt5a. Wnt5a is a member of the Wnt family of proteins, which play an important role during development and tumorigenesis. In melanoma, Wnt5a signaling is mediated by an autocrine loop that also modulates the activity of midkine. Midkine is a 16kDa protein that is elevated in many different cancers. Overexpression of midkine promotes the growth, migration, survival and angiogenic capability of the tumors. However, the molecular mechanisms that regulate the activity of midkine are unknown, especially in metastatic melanoma. We asked whether Wnt5a can positively regulate the functions of midkine to promote the progression of metastatic melanoma. To address this, we analyzed melanoma cells for midkine expression and observed that Wnt5a high melanoma cells also express high levels of midkine. Further, an increase in Wnt5a signaling by treatment with recombinant Wnt5a led to an increased midkine signaling. By performing 3D spheroid assays, we observed that higher expression of midkine is correlated with an increased metastatic ability of the cells. The expression of midkine also correlated with an increased resistance to BRAF inhibitors in BRAF V600E mutated melanoma. This indicates that midkine may signal through MAPK pathway to affect invasion and drug resistance in metastatic melanoma. Developing adjuvant therapies targeting midkine can prolong the overall survival of patients with metastatic melanoma. Citation Format: Amanpreet Kaur, Michael O9Connell, Marie Webster, Reeti Behera, Vanessa Dang, Abibatou Ndoye, Ashani Weeraratna. Midkine as a potential target for combating drug resistance and invasion in melanoma. [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 5092. doi:10.1158/1538-7445.AM2015-5092