Expression of versican isoforms V0/V1 by pancreatic cancer associated fibroblasts increases fibroblast proliferation.

BACKGROUND: Versican is a large extracellular matrix (ECM) proteoglycan with four isoforms V0-3. Elevated V0/V1 levels in breast cancer and glioma regulate cell migration and proliferation, but the role of versican in pancreatic ductal adenocarcinoma (PDAC) remains unclear. METHODS: In this study, we evaluated the expression levels of versican isoforms, as well as their cellular source and interacting partners, in vivo, in human and mouse primary and metastatic PDAC tumours and in vitro, in pancreatic tumour cells and fibroblasts using immunostaining, confocal microscopy and qPCR techniques. We also investigated the effect of versican expression on fibroblast proliferation and migration using genetic and pharmacological approaches. RESULTS: We found that versican V0/V1 is highly expressed by cancer-associated fibroblasts (CAFs) in mouse and human primary and metastatic PDAC tumours. Our data also show that exposing fibroblasts to tumour-conditioned media upregulates V0 and V1 expressions, while Verbascoside (a CD44 inhibitor) downregulates V0/V1 expression. Importantly, V0/V1 knockdown significantly inhibits fibroblast proliferation. Mechanistically, we found that inhibiting hyaluronan synthesis does not affect versican co-localisation with CD44 in fibroblasts. CONCLUSION: CAFs express high levels of versican V0/V1 in primary and liver metastatic PDAC tumours and versican V0/V1 supports fibroblast proliferation.

Chemotherapy-induced infiltration of neutrophils promotes pancreatic cancer metastasis via Gas6/AXL signalling axis.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease and cytotoxic chemotherapy is the standard of care treatment for patients with advanced disease. Here, we investigate how the microenvironment in PDAC liver metastases reacts to chemotherapy and its role in metastatic disease progression post-treatment, an area which is poorly understood. DESIGN: The impact of chemotherapy on metastatic disease progression and immune cell infiltrates was characterised using flow and mass cytometry combined with transcriptional and histopathological analysis in experimental PDAC liver metastases mouse models. Findings were validated in patient derived liver metastases and in an autochthonous PDAC mouse model. Human and murine primary cell cocultures and ex vivo patient-derived liver explants were deployed to gain mechanistical insights on whether and how chemotherapy affects the metastatic tumour microenvironment. RESULTS: We show that in vivo, chemotherapy induces an initial infiltration of proinflammatory macrophages into the liver and activates cytotoxic T cells, leading only to a temporary restraining of metastatic disease progression. However, after stopping treatment, neutrophils are recruited to the metastatic liver via CXCL1 and 2 secretion by metastatic tumour cells. These neutrophils express growth arrest specific 6 (Gas6) which leads to AXL receptor activation on tumour cells enabling their regrowth. Disruption of neutrophil infiltration or inhibition of the Gas6/AXL signalling axis in combination with chemotherapy inhibits metastatic growth. Chemotherapy increases Gas6 expression in circulating neutrophils from patients with metastatic pancreatic cancer and recombinant Gas6 is sufficient to promote tumour cell proliferation ex vivo, in patient-derived metastatic liver explants. CONCLUSION: Combining chemotherapy with Gas6/AXL or neutrophil targeted therapy could provide a therapeutic benefit for patients with metastatic pancreatic cancer.

Mesothelin Secretion by Pancreatic Cancer Cells Co-opts Macrophages and Promotes Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease, yet effective treatments to inhibit PDAC metastasis are lacking. The rich PDAC tumor microenvironment plays a major role in disease progression. Macrophages are the most abundant immune cell population in PDAC tumors and can acquire a range of functions that either hinder or promote tumor growth and metastasis. Here, we identified that mesothelin secretion by pancreatic cancer cells co-opts macrophages to support tumor growth and metastasis of cancer cells to the lungs, liver, and lymph nodes. Mechanistically, secretion of high levels of mesothelin by metastatic cancer cells induced the expression of VEGF alpha (VEGFA) and S100A9 in macrophages. Macrophage-derived VEGFA fed back to cancer cells to support tumor growth, and S100A9 increased neutrophil lung infiltration and formation of neutrophil extracellular traps. These results reveal a role for mesothelin in regulating macrophage functions and interaction with neutrophils to support PDAC metastasis. SIGNIFICANCE: Mesothelin secretion by cancer cells supports pancreatic cancer metastasis by inducing macrophage secretion of VEGFA and S100A9 to support cancer cell proliferation and survival, recruit neutrophils, and stimulate neutrophil extracellular trap formation. See related commentary by Alewine, p. 513.

Macrophage-fibroblast JAK/STAT dependent crosstalk promotes liver metastatic outgrowth in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease for which better therapies are urgently needed. Fibroblasts and macrophages are heterogeneous cell populations able to enhance metastasis, but the role of a macrophage-fibroblast crosstalk in regulating their pro-metastatic functions remains poorly understood. Here we deconvolve how macrophages regulate metastasis-associated fibroblast (MAF) heterogeneity in the liver. We identify three functionally distinct MAF populations, among which the generation of pro-metastatic and immunoregulatory myofibroblastic-MAFs (myMAFs) critically depends on macrophages. Mechanistically, myMAFs are induced through a STAT3-dependent mechanism driven by macrophage-derived progranulin and cancer cell-secreted leukaemia inhibitory factor (LIF). In a reciprocal manner, myMAF secreted osteopontin promotes an immunosuppressive macrophage phenotype resulting in the inhibition of cytotoxic T cell functions. Pharmacological blockade of STAT3 or myMAF-specific genetic depletion of STAT3 restores an anti-tumour immune response and reduces metastases. Our findings provide molecular insights into the complex macrophage-fibroblast interactions in tumours and reveal potential targets to inhibit PDAC liver metastasis.

Efferocytosis reprograms the tumor microenvironment to promote pancreatic cancer liver metastasis.

Pancreatic ductal adenocarcinoma is a highly metastatic disease and macrophages support liver metastases. Efferocytosis, or engulfment of apoptotic cells by macrophages, is an essential process in tissue homeostasis and wound healing, but its role in metastasis is less well understood. Here, we found that the colonization of the hepatic metastatic site is accompanied by low-grade tissue injury and that efferocytosis-mediated clearance of parenchymal dead cells promotes macrophage reprogramming and liver metastasis. Mechanistically, progranulin expression in macrophages is necessary for efficient efferocytosis by controlling lysosomal acidification via cystic fibrosis transmembrane conductance regulator and the degradation of lysosomal cargo, resulting in LXRα/RXRα-mediated macrophage conversion and upregulation of arginase 1. Pharmacological blockade of efferocytosis or macrophage-specific genetic depletion of progranulin impairs macrophage conversion, improves CD8+ T cell functions, and reduces liver metastasis. Our findings reveal how hard-wired functions of macrophages in tissue repair contribute to liver metastasis and identify potential targets for prevention of pancreatic ductal adenocarcinoma liver metastasis.

RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia.

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Caspase-8 isoform 6 promotes death effector filament formation independent of microtubules.

Caspase-8 can trigger cell death following prodomain-mediated recruitment to the 'death-inducing signaling complex.' The prodomain consists of two death effector domain (DED) motifs that undergo homotypic interactions within the cell. Aside from mediating recruitment of procaspase-8, the prodomains have also been implicated in regulating cell survival, proliferation, death, senescence, differentiation, and substrate attachment. Here, we perform the initial characterization of a novel isoform of caspase-8, designated caspase-8 isoform 6 (Casp-8.6), which encodes both prodomain DEDs followed by a unique C-terminal tail. Casp-8.6 is detected in cells of the hematopoietic compartment as well as several other tissues. When Casp-8.6 expression is reconstituted in caspase-8-deficient cells, Casp-8.6 does not significantly impact cellular proliferation, contrasting with our previous results using a domain-defined 'DED-only' construct that lacks the C-terminal tail. Like the DED-only construct, Casp-8.6 also robustly forms 'death effector' filaments, but in contrast to the DED construct, it does not exhibit a dependence upon intact microtubules to scaffold filament formation. Both types of death effector filaments promote apoptosis when expressed in the presence of full length caspase-8 (isoform 1). Together, the results implicate Casp-8.6 as a new physiological modulator of apoptosis.

The marine lipopeptide somocystinamide A triggers apoptosis via caspase 8.

Screening for novel anticancer drugs in chemical libraries isolated from marine organisms, we identified the lipopeptide somocystinamide A (ScA) as a pluripotent inhibitor of angiogenesis and tumor cell proliferation. The antiproliferative activity was largely attributable to induction of programmed cell death. Sensitivity to ScA was significantly increased among cells expressing caspase 8, whereas siRNA knockdown of caspase 8 increased survival after exposure to ScA. ScA rapidly and efficiently partitioned into liposomes while retaining full antiproliferative activity. Consistent with the induction of apoptosis via the lipid compartment, we noted accumulation and aggregation of ceramide in treated cells and subsequent colocalization with caspase 8. Angiogenic endothelial cells were extremely sensitive to ScA. Picomolar concentrations of ScA disrupted proliferation and endothelial tubule formation in vitro. Systemic treatment of zebrafish or local treatment of the chick chorioallantoic membrane with ScA resulted in dose-dependent inhibition of angiogenesis, whereas topical treatment blocked tumor growth among caspase-8-expressing tumors. Together, the results reveal an unexpected mechanism of action for this unique lipopeptide and suggest future development of this and similar agents as antiangiogenesis and anticancer drugs.

Cancer-Associated Fibroblast Mediated Inhibition of CD8+ Cytotoxic T Cell Accumulation in Tumours: Mechanisms and Therapeutic Opportunities.

The tumour microenvironment (TME) is the complex environment in which various non-cancerous stromal cell populations co-exist, co-evolve and interact with tumour cells, having a profound impact on the progression of solid tumours. The TME is comprised of various extracellular matrix (ECM) proteins in addition to a variety of immune and stromal cells. These include tumour-associated macrophages, regulatory T cells (Tregs), myeloid-derived suppressor cells, as well as endothelial cells, pericytes and cancer-associated fibroblasts (CAFs). CAFs are the most abundant stromal cell population in many tumours and support cancer progression, metastasis and resistance to therapies through bidirectional signalling with both tumour cells and other cells within the TME. More recently, CAFs have been shown to also affect the anti-tumour immune response through direct and indirect interactions with immune cells. In this review, we specifically focus on the interactions between CAFs and cytotoxic CD8+ T cells, and on how these interactions affect T cell recruitment, infiltration and function in the tumour. We additionally provide insight into the therapeutic implications of targeting these interactions, particularly in the context of cancer immunotherapy.

Liver Tropism in Cancer: The Hepatic Metastatic Niche.

The liver is the largest organ in the human body and is prone for cancer metastasis. Although the metastatic pattern can differ depending on the cancer type, the liver is the organ to which cancer cells most frequently metastasize for the majority of prevalent malignancies. The liver is unique in several aspects: the vascular structure is highly permeable and has unparalleled dual blood connectivity, and the hepatic tissue microenvironment presents a natural soil for the seeding of disseminated tumor cells. Although 70% of the liver is composed of the parenchymal hepatocytes, the remaining 30% is composed of nonparenchymal cells including Kupffer cells, liver sinusoidal endothelial cells, and hepatic stellate cells. Recent discoveries show that both the parenchymal and the nonparenchymal cells can modulate each step of the hepatic metastatic cascade, including the initial seeding and colonization as well as the decision to undergo dormancy versus outgrowth. Thus, a better understanding of the molecular mechanisms orchestrating the formation of a hospitable hepatic metastatic niche and the identification of the drivers supporting this process is critical for the development of better therapies to stop or at least decrease liver metastasis. The focus of this perspective is on the bidirectional interactions between the disseminated cancer cells and the unique hepatic metastatic niche.

Blockade of Stromal Gas6 Alters Cancer Cell Plasticity, Activates NK Cells, and Inhibits Pancreatic Cancer Metastasis.

Pancreatic ductal adenocarcinoma (PDA) is one of the deadliest cancers due to its aggressive and metastatic nature. PDA is characterized by a rich tumor stroma with abundant macrophages, fibroblasts, and collagen deposition that can represent up to 90% of the tumor mass. Activation of the tyrosine kinase receptor AXL and expression of its ligand growth arrest-specific protein 6 (Gas6) correlate with a poor prognosis and increased metastasis in pancreatic cancer patients. Gas6 is a multifunctional protein that can be secreted by several cell types and regulates multiple processes, including cancer cell plasticity, angiogenesis, and immune cell functions. However, the role of Gas6 in pancreatic cancer metastasis has not been fully investigated. In these studies we find that, in pancreatic tumors, Gas6 is mainly produced by tumor associated macrophages (TAMs) and cancer associated fibroblasts (CAFs) and that pharmacological blockade of Gas6 signaling partially reverses epithelial-to-mesenchymal transition (EMT) of tumor cells and supports NK cell activation, thereby inhibiting pancreatic cancer metastasis. Our data suggest that Gas6 simultaneously acts on both the tumor cells and the NK cells to support pancreatic cancer metastasis. This study supports the rationale for targeting Gas6 in pancreatic cancer and use of NK cells as a potential biomarker for response to anti-Gas6 therapy.

Apoptosis and pathogenesis of melanoma and nonmelanoma skin cancer.

Skin cancers, i.e., basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and melanoma, belong to the most frequent tumors. Their formation is based on constitutional and/or inherited factors usually combined with environmental factors, mainly UV-irradiation through long term sun exposure. UV-light can randomly induce DNA damage in keratinocytes, but it can also mutate genes essential for control and surveillance in the skin epidermis. Various repair and safety mechanisms exist to maintain the integrity of the skin epidermis. For example, UV-light damaged DNA is repaired and if this is not possible, the DNA damaged cells are eliminated by apoptosis (sunburn cells). This occurs under the control of the p53 suppressor gene. Fas-ligand (FasL), a member of the tumor necrosis superfamily, which is preferentially expressed in the basal layer of the skin epidermis, is a key surveillance molecule involved in the elimination of sunburn cells, but also in the prevention of cell transformation. However, UV light exposure downregulates FasL expression in keratinocytes and melanocytes leading to the loss of its sensor function. This increases the risk that transformed cells are not eliminated anymore. Moreover, important control and surveillance genes can also be directly affected by UV-light. Mutation in the p53 gene is the starting point for the formation of SCC and some forms of BCC. Other BCCs originate through UV light mediated mutations of genes of the hedgehog signaling pathway which are essential for the maintainance of cell growth and differentiation. The transcription factor Gli2 plays a key role within this pathway, indeed, Gli2 is responsible for the marked apoptosis resistance of the BCCs. The formation of malignant melanoma is very complex. Melanocytes form nevi and from the nevi melanoma can develop through mutations in various genes. Once the keratinocytes or melanocytes have been transformed they re-express FasL which may allow the expanding tumor to evade the attack of immune effector cells. FasL which is involved in immune evasion or genes which govern the apoptosis resistance, e.g., Gli2 could therefore be prime targets to prevent tumor formation and growth. Attempts to silence these genes by RNA interference using gene specific short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) have been functionally successful not only in tissue cultures and tumor tissues, but also in a mouse model. Thus, siRNAs and/or shRNAs may become a novel and promising approach to treat skin cancers at an early stage.

Macrophages and Fibroblasts, Key Players in Cancer Chemoresistance.

Chemotherapy is routinely used in cancer treatment to eliminate primary and metastatic tumor cells. However, tumors often display or develop resistance to chemotherapy. Mechanisms of chemoresistance can be either tumor cell autonomous or mediated by the tumor surrounding non-malignant cells, also known as stromal cells, which include fibroblasts, immune cells, and cells from the vasculature. Therapies targeting cancer cells have shown limited effectiveness in tumors characterized by a rich tumor stroma. Tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs) are the most abundant non-cancerous cells in the tumor stroma and have emerged as key players in cancer progression, metastasis and resistance to therapies. This review describes the recent advances in our understanding of how CAFs and TAMs confer chemoresistance to tumor cells and discusses the therapeutic opportunities of combining anti-tumor with anti-stromal therapies. The continued elucidation of the mechanisms by which TAMs and CAFs mediate resistance to therapies will allow the development of improved combination treatments for cancer patients.

Blockade of insulin-like growth factors increases efficacy of paclitaxel in metastatic breast cancer.

Breast cancer remains the leading cause of cancer death in women owing to metastasis and the development of resistance to established therapies. Macrophages are the most abundant immune cells in the breast tumor microenvironment and can both inhibit and support cancer progression. Thus, gaining a better understanding of how macrophages support cancer could lead to the development of more effective therapies. In this study, we find that breast cancer-associated macrophages express high levels of insulin-like growth factors 1 and 2 (IGFs) and are the main source of IGFs within both primary and metastatic tumors. In total, 75% of breast cancer patients show activation of insulin/IGF-1 receptor signaling and this correlates with increased macrophage infiltration and advanced tumor stage. In patients with invasive breast cancer, activation of Insulin/IGF-1 receptors increased to 87%. Blocking IGF in combination with paclitaxel, a chemotherapeutic agent commonly used to treat breast cancer, showed a significant reduction in tumor cell proliferation and lung metastasis in pre-clinical breast cancer models compared to paclitaxel monotherapy. Our findings provide the rationale for further developing the combination of paclitaxel with IGF blockers for the treatment of invasive breast cancer, and Insulin/IGF1R activation and IGF+ stroma cells as potential biomarker candidates for further evaluation.

Macrophage-Derived Granulin Drives Resistance to Immune Checkpoint Inhibition in Metastatic Pancreatic Cancer.

The ability of disseminated cancer cells to evade the immune response is a critical step for efficient metastatic progression. Protection against an immune attack is often provided by the tumor microenvironment that suppresses and excludes cytotoxic CD8+ T cells. Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive metastatic disease with unmet needs, yet the immunoprotective role of the metastatic tumor microenvironment in pancreatic cancer is not completely understood. In this study, we find that macrophage-derived granulin contributes to cytotoxic CD8+ T-cell exclusion in metastatic livers. Granulin expression by macrophages was induced in response to colony-stimulating factor 1. Genetic depletion of granulin reduced the formation of a fibrotic stroma, thereby allowing T-cell entry at the metastatic site. Although metastatic PDAC tumors are largely resistant to anti-PD-1 therapy, blockade of PD-1 in granulin-depleted tumors restored the antitumor immune defense and dramatically decreased metastatic tumor burden. These findings suggest that targeting granulin may serve as a potential therapeutic strategy to restore CD8+ T-cell infiltration in metastatic PDAC, thereby converting PDAC metastatic tumors, which are refractory to immune checkpoint inhibitors, into tumors that respond to immune checkpoint inhibition therapies.Significance: These findings uncover a mechanism by which metastatic PDAC tumors evade the immune response and provide the rationale for targeting granulin in combination with immune checkpoint inhibitors for the treatment of metastatic PDAC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/15/4253/F1.large.jpg Cancer Res; 78(15); 4253-69. ©2018 AACR.

Blockade of MIF-CD74 Signalling on Macrophages and Dendritic Cells Restores the Antitumour Immune Response Against Metastatic Melanoma.

Mounting an effective immune response against cancer requires the activation of innate and adaptive immune cells. Metastatic melanoma is the most aggressive form of skin cancer. While immunotherapies have shown a remarkable success in melanoma treatment, patients develop resistance by mechanisms that include the establishment of an immune suppressive tumor microenvironment. Thus, understanding how metastatic melanoma cells suppress the immune system is vital to develop effective immunotherapies against this disease. In this study, we find that macrophages (MOs) and dendritic cells (DCs) are suppressed in metastatic melanoma and that the Ig-CDR-based peptide C36L1 is able to restore MOs and DCs' antitumorigenic and immunogenic functions and to inhibit metastatic growth in lungs. Specifically, C36L1 treatment is able to repolarize M2-like immunosuppressive MOs into M1-like antitumorigenic MOs, and increase the number of immunogenic DCs, and activated cytotoxic T cells, while reducing the number of regulatory T cells and monocytic myeloid-derived suppressor cells in metastatic lungs. Mechanistically, we find that C36L1 directly binds to the MIF receptor CD74 which is expressed on MOs and DCs, disturbing CD74 structural dynamics and inhibiting MIF signaling on these cells. Interfering with MIF-CD74 signaling on MOs and DCs leads to a decrease in the expression of immunosuppressive factors from MOs and an increase in the capacity of DCs to activate cytotoxic T cells. Our findings suggest that interfering with MIF-CD74 immunosuppressive signaling in MOs and DCs, using peptide-based immunotherapy can restore the antitumor immune response in metastatic melanoma. Our study provides the rationale for further development of peptide-based therapies to restore the antitumor immune response in metastatic melanoma.

Chemoresistance in Pancreatic Cancer Is Driven by Stroma-Derived Insulin-Like Growth Factors.

Tumor-associated macrophages (TAM) and myofibroblasts are key drivers in cancer that are associated with drug resistance in many cancers, including pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the molecular mechanisms by which TAM and fibroblasts contribute to chemoresistance is unclear. In this study, we found that TAM and myofibroblasts directly support chemoresistance of pancreatic cancer cells by secreting insulin-like growth factors (IGF) 1 and 2, which activate insulin/IGF receptors on pancreatic cancer cells. Immunohistochemical analysis of biopsies from patients with pancreatic cancer revealed that 72% of the patients expressed activated insulin/IGF receptors on tumor cells, and this positively correlates with increased CD163+ TAM infiltration. In vivo, we found that TAM and myofibroblasts were the main sources of IGF production, and pharmacologic blockade of IGF sensitized pancreatic tumors to gemcitabine. These findings suggest that inhibition of IGF in combination with chemotherapy could benefit patients with PDAC, and that insulin/IGF1R activation may be used as a biomarker to identify patients for such therapeutic intervention. Cancer Res; 76(23); 6851-63. ©2016 AACR.

Macrophage-secreted granulin supports pancreatic cancer metastasis by inducing liver fibrosis.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating metastatic disease for which better therapies are urgently needed. Macrophages enhance metastasis in many cancer types; however, the role of macrophages in PDAC liver metastasis remains poorly understood. Here we found that PDAC liver metastasis critically depends on the early recruitment of granulin-secreting inflammatory monocytes to the liver. Mechanistically, we demonstrate that granulin secretion by metastasis-associated macrophages (MAMs) activates resident hepatic stellate cells (hStCs) into myofibroblasts that secrete periostin, resulting in a fibrotic microenvironment that sustains metastatic tumour growth. Disruption of MAM recruitment or genetic depletion of granulin reduced hStC activation and liver metastasis. Interestingly, we found that circulating monocytes and hepatic MAMs in PDAC patients express high levels of granulin. These findings suggest that recruitment of granulin-expressing inflammatory monocytes plays a key role in PDAC metastasis and may serve as a potential therapeutic target for PDAC liver metastasis.