Although most melanoma cases may be treated by surgical intervention upon early diagnosis, a significant portion of patients can still be refractory, presenting low survival rates within 5 years after the discovery of the illness. As a hallmark, melanomas are highly prone to evolve into metastatic sites. Moreover, melanoma tumors are highly resistant to most available drug therapies and their incidence have increased over the years, therefore leading to public health concerns about the development of novel therapies. Therefore, researches are getting deeper in unveiling the mechanisms by which melanoma initiation can be triggered and sustained. In this context, important progress has been achieved regarding the roles and the impact of cellular signaling pathways in melanoma. This knowledge has provided tools for the development of therapies based on the intervention of signal(s) promoted by these cascades. In this review, we summarize the importance of major signaling pathways (mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase (PI3K)-Akt, Wnt, nuclear factor κ-light-chain-enhancer of activated B cell (NF-κB), Janus kinase (JAK)-signal transducer and activator of transcription (STAT), transforming growth factor β (TGF-β) and Notch) in skin homeostasis and melanoma progression. Available and developing melanoma therapies interfering with these signaling cascades are further discussed.

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The tumor microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumorigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumor growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple-negative breast cancers and that it directly induces Cancer-associated fibroblasts phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g., CXCL1), cytokines (e.g., IL-6) and growth factors (e.g., PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

AbstractThe tumour microenvironment (TME) is an important mediator of breast cancer progression. Cancer-associated fibroblasts (CAFs) constitute a major component of the TME and may originate from tissue-associated fibroblasts or infiltrating mesenchymal stromal cells (MSCs). The mechanisms by which cancer cells activate fibroblasts and recruit MSCs to the TME are largely unknown, but likely include deposition of a pro-tumourigenic secretome. The secreted embryonic protein NODAL is clinically associated with breast cancer stage and promotes tumour growth, metastasis, and vascularization. Herein, we show that NODAL expression correlates with the presence of activated fibroblasts in human triple negative breast cancers and that it directly induces CAF phenotypes. We further show that NODAL reprograms cancer cell secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), cytokines (e.g. IL-6) and growth factors (e.g. PDGFRA), leading to alterations in MSC chemotaxis. We therefore demonstrate a hitherto unappreciated mechanism underlying the dynamic regulation of the TME.

Background: Despite the increase in patients' survival over the last years, multiple myeloma (MM) remains incurable, being persistence of cancer stem cells (CSCs) a probable cause of drug resistance and disease relapse. It is possible to isolate these cells using surface antigen expression pattern (CD19+/CD34+/CD138-) and the activity of an enzyme from aldehyde dehydrogenase (ALDH) family (Boucher et al., 2012). We believe that using CD19 as potential marker of MM-CSCs makes CAR-T cell therapy against CD19 an option to eradicate residual MM disease. Aims: To isolate and characterize immunophenotypically, functionally and by gene expression the MM-CSCs derived from bone marrow (BM) samples of newly-diagnosed MM patients, focusing on identification of possible therapeutic targets. Methods: BM aspirates were collected and CD138+ cells were separated by magnetic sorting. The remaining cells were submitted to sorting by flow cytometry on FACSAria II (Becton, Dickinson and Company, Franklin Lakes, NJ, USA), labeled with anti-CD19 Pacific Blue (Invitrogen, Carlsbad, CA, USA), anti-CD34 PE Cy7 and anti-CD138 APC (both from Becton, Dickinson and Company, Franklin Lakes, NJ, USA) antibodies, in addition to Aldefluor™ reagent (StemCell Technology, Vancouver, British Columbia, Canada). RNA was extracted and pre-amplified for PCR array analysis using the RT² Profiler™ PCR Array Human Cancer Stem Cells(Qiagen, Hilden, Germany) to assess the expression profile of 84 genes related to cancer stem cells, and the results were evaluated with the online software provided by the platform manufacturer. Results: MM-CSCs (CD34+/CD19+/CD138-/ALDH1+) were isolated by flow cytometry from MM samples and presented median of 1,748.5 events (ranging from 56 to 16,633, n = 16). For comparison purposes, CD138+ MM tumor cells were isolated and used as "control group" (median of events 72,904, ranging from 1,536 to 312,504, n = 15). RNA from 16 MM-CSC samples and 6 controls were analyzed by qPCR. Considering 2-ΔCt calculation (GAPDH as normalizer) and fold change of 2, 11 genes were considered differentially expressed in MM-CSCs when compared to tumor plasma cells (p<0.05) (4 overexpressed and 7 underexpressed), as calculated by Student's T-test. Overexpression of CD38 in the control group confirms the origin of tumor plasma cells. Other genes overexpressed in tumor plasma cells include ITGA6 and PECAM1 (adhesion-related molecules); MERTK (signal transduction); BMI1; DKK1 (Wnt signaling pathway), and ID1 (cell migration and metastasis). Among the MM-CSCs overexpressed genes, ALDH1A1, ENG and ITGB1 are CSC markers and are related to stem cell functions; and WEE1 is a cell cycle-related gene which regulates G2-M checkpoint and promotes cell cycles arrest for further DNA repair. Discussion and Conclusions: Our preliminary data shows that cells isolated using Boucher et al. (2012) selected markers have MM-CSCs characteristics, validated by the expression of at least three genes related to CSCs: ALDH1A1, ENG and ITGB1. We also identified WEE1overexpression in MM-CSCs and this data is a new finding in MM studies. WEE1 overexpression has been described in several cancers, such as mantle cell lymphoma, colorectal cancer, breast cancer and glioblastoma. WEE1 inhibition by siRNAs or small molecule inhibitors AZD1775 (MK1775) and PD0407824, as shown in preclinical and clinical trials, impairs G2-M arrest and promotes premature entry in mitosis, leading to consequent cell death if DNA damage is irreparable (Mueller & Haas-Kogan, 2015). Therefore, these inhibitors could also be explored in MM treatment. Furthermore, we suggest that CAR-T cell therapy against CD19 might be and option to eradicate residual MM disease, since we confirmed that this antigen is present in MM-CSCs surface. Financial support: FAPESP 2010/17668-6 and FAPESP 2015/23983-5. Ethical approval: 0127/2014. Disclaimer: Oliveira MB and Dantonio PM equally contributed to this abstract. Disclosures No relevant conflicts of interest to declare.

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The tumour microenvironment (TME), consisting of stromal cell types including fibroblasts, endothelial, immune and mesenchymal stromal cells (MSCs), is an important mediator of breast cancer (BCa) progression. BCa cells regulate the composition of the TME by secreting a myriad of factors; however, mechanisms governing the dynamic reciprocity between BCa and TME components are largely unknown. Cancer-associated fibroblasts (CAFs) are a major stromal component in many BCa and have been shown to be activated by BCa cells. MSCs recruited to the TME are an important source of CAFs in many cancers; therefore, characterizing the factors involved in MSC recruitment could provide additional avenues for therapeutic intervention by disrupting the TME in BCa. NODAL, an embryonic morphogen belonging to the TGF-beta superfamily, is clinically associated with BCa progression, and has been shown to sustain BCa stem cells and to promote tumour growth, metastasis, and vascularization. Herein, we report that NODAL expression positively correlates with the presence of CAFs in the stroma of triple-negative human BCa tissues, as determined by NODAL and a-smooth muscle Actin (SMA) staining. Further, our in vitro studies show that NODAL directly induces primary fibroblast activation and chemotaxis. Proteomic analysis of the conditioned media of NODAL-overexpressing MDA-MB-231 and SUM149 BCa cells by mass spectrometry reveal that NODAL reprograms BCa secretomes by simultaneously altering levels of chemokines (e.g. CXCL1), growth factors (e.g. PDGFRA) and cytokines (e.g. IL-6), and that the NODAL-regulated secretome, but not NODAL directly, impacts the ability of BCa cells to affect MSC chemotaxis. We provide evidence that IL-6 promotes MSC chemotaxis, and further discuss the context-dependent effects of NODAL in BCa cell lines. Collectively, our results suggest that factors present in NODAL-regulated secretomes may induce MSC recruitment to the breast TME, where they may contribute to CAF population, therefore demonstrating a hitherto unappreciated role of NODAL as a dynamic regulator of breast TME components. Citation Format: Dylan Dieters-Castator, Paola M. Dantonio, Matt Piaseczny, Guihua Zhang, Jiahui Liu, Miljan Kuljanin, Stephen Sherman, Michael Jewer, Katherine Quesnel, Eun-Young Kang, Martin Köbel, Gabrielle M. Siegers, Andrew Leask, David Hess, Gilles Lajoie, Lynne-Marie Postovit. Embryonic protein NODAL as a potential modulator of the tumour microenvironment: Breast cancer secretome reprogramming and fibroblast activation [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr PO028.

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