Macrophages direct cancer cells through a LOXL2-mediated metastatic cascade in pancreatic ductal adenocarcinoma.

OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.

Dysregulated splicing factor SF3B1 unveils a dual therapeutic vulnerability to target pancreatic cancer cells and cancer stem cells with an anti-splicing drug.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, requiring novel treatments to target both cancer cells and cancer stem cells (CSCs). Altered splicing is emerging as both a novel cancer hallmark and an attractive therapeutic target. The core splicing factor SF3B1 is heavily altered in cancer and can be inhibited by Pladienolide-B, but its actionability in PDAC is unknown. We explored the presence and role of SF3B1 in PDAC and interrogated its potential as an actionable target. METHODS: SF3B1 was analyzed in PDAC tissues, an RNA-seq dataset, and publicly available databases, examining associations with splicing alterations and key features/genes. Functional assays in PDAC cell lines and PDX-derived CSCs served to test Pladienolide-B treatment effects in vitro, and in vivo in zebrafish and mice. RESULTS: SF3B1 was overexpressed in human PDAC and associated with tumor grade and lymph-node involvement. SF3B1 levels closely associated with distinct splicing event profiles and expression of key PDAC players (KRAS, TP53). In PDAC cells, Pladienolide-B increased apoptosis and decreased multiple tumor-related features, including cell proliferation, migration, and colony/sphere formation, altering AKT and JNK signaling, and favoring proapoptotic splicing variants (BCL-XS/BCL-XL, KRASa/KRAS, Δ133TP53/TP53). Importantly, Pladienolide-B similarly impaired CSCs, reducing their stemness capacity and increasing their sensitivity to chemotherapy. Pladienolide-B also reduced PDAC/CSCs xenograft tumor growth in vivo in zebrafish and in mice. CONCLUSION: SF3B1 overexpression represents a therapeutic vulnerability in PDAC, as altered splicing can be targeted with Pladienolide-B both in cancer cells and CSCs, paving the way for novel therapies for this lethal cancer.

The CXCL12 Crossroads in Cancer Stem Cells and Their Niche.

Cancer stem cells (CSCs) are defined as a subpopulation of "stem"-like cells within the tumor with unique characteristics that allow them to maintain tumor growth, escape standard anti-tumor therapies and drive subsequent repopulation of the tumor. This is the result of their intrinsic "stem"-like features and the strong driving influence of the CSC niche, a subcompartment within the tumor microenvironment that includes a diverse group of cells focused on maintaining and supporting the CSC. CXCL12 is a chemokine that plays a crucial role in hematopoietic stem cell support and has been extensively reported to be involved in several cancer-related processes. In this review, we will provide the latest evidence about the interactions between CSC niche-derived CXCL12 and its receptors-CXCR4 and CXCR7-present on CSC populations across different tumor entities. The interactions facilitated by CXCL12/CXCR4/CXCR7 axes seem to be strongly linked to CSC "stem"-like features, tumor progression, and metastasis promotion. Altogether, this suggests a role for CXCL12 and its receptors in the maintenance of CSCs and the components of their niche. Moreover, we will also provide an update of the therapeutic options being currently tested to disrupt the CXCL12 axes in order to target, directly or indirectly, the CSC subpopulation.

Exploiting oxidative phosphorylation to promote the stem and immunoevasive properties of pancreatic cancer stem cells.

Pancreatic ductal adenocarcinoma (PDAC), the fourth leading cause of cancer death, has a 5-year survival rate of approximately 7-9%. The ineffectiveness of anti-PDAC therapies is believed to be due to the existence of a subpopulation of tumor cells known as cancer stem cells (CSCs), which are functionally plastic, and have exclusive tumorigenic, chemoresistant and metastatic capacities. Herein, we describe a 2D in vitro system for long-term enrichment of pancreatic CSCs that is amenable to biological and CSC-specific studies. By changing the carbon source from glucose to galactose in vitro, we force PDAC cells to utilize OXPHOS, resulting in enrichment of CSCs defined by increased CSC biomarker and pluripotency gene expression, greater tumorigenic potential, induced but reversible quiescence, increased OXPHOS activity, enhanced invasiveness, and upregulated immune evasion properties. This CSC enrichment method can facilitate the discovery of new CSC-specific hallmarks for future development into targets for PDAC-based therapies.

ISG15 and ISGylation is required for pancreatic cancer stem cell mitophagy and metabolic plasticity.

Pancreatic cancer stem cells (PaCSCs) drive pancreatic cancer tumorigenesis, chemoresistance and metastasis. While eliminating this subpopulation of cells would theoretically result in tumor eradication, PaCSCs are extremely plastic and can successfully adapt to targeted therapies. In this study, we demonstrate that PaCSCs increase expression of interferon-stimulated gene 15 (ISG15) and protein ISGylation, which are essential for maintaining their metabolic plasticity. CRISPR-mediated ISG15 genomic editing reduces overall ISGylation, impairing PaCSCs self-renewal and their in vivo tumorigenic capacity. At the molecular level, ISG15 loss results in decreased mitochondrial ISGylation concomitant with increased accumulation of dysfunctional mitochondria, reduced oxidative phosphorylation (OXPHOS) and impaired mitophagy. Importantly, disruption in mitochondrial metabolism affects PaCSC metabolic plasticity, making them susceptible to prolonged inhibition with metformin in vivo. Thus, ISGylation is critical for optimal and efficient OXPHOS by ensuring the recycling of dysfunctional mitochondria, and when absent, a dysregulation in mitophagy occurs that negatively impacts PaCSC stemness.

The Interactions Between Cancer Stem Cells and the Innate Interferon Signaling Pathway.

Interferons (IFNs) form a family of cytokines with pleiotropic effects that modulate the immune response against multiple challenges like viral infections, autoimmune diseases, and cancer. While numerous anti-tumor activities have been described for IFNs, IFNs have also been associated with tumor growth and progression. The effect of IFNs on apoptosis, angiogenesis, tumor cell immunogenicity, and modulation of immune cells have been largely studied; however, less is known about their specific effects on cancer stem cells (CSCs). CSCs constitute a subpopulation of tumor cells endowed with stem-like properties including self-renewal, chemoresistance, tumorigenic capacity, and quiescence. This rare and unique subpopulation of cells is believed to be responsible for tumor maintenance, metastatic spread, and relapse. Thus, this review aims to summarize and discuss the current knowledge of the anti- and pro-CSCs effects of IFNs and also to highlight the need for further research on the interplay between IFNs and CSCs. Importantly, understanding this interplay will surely help to exploit the anti-tumor effects of IFNs, specifically those that target CSCs.

Tumor-associated macrophage-secreted 14-3-3ζ signals via AXL to promote pancreatic cancer chemoresistance.

Pancreatic ductal adenocarcinoma (PDAC) is an inherently chemoresistant tumor. Chemotherapy leads to apoptosis of cancer cells, and in previous studies we have shown that tumor-associated macrophage (TAM) infiltration increases following chemotherapy in PDAC. Since one of the main functions of macrophages is to eliminate apoptotic cells, we hypothesized that TAMs phagocytose chemotherapy-induced apoptotic cells and secrete factors, which favor PDAC chemoresistance. To test this hypothesis, primary human PDAC cultures were treated with conditioned media (CM) from monocyte-derived macrophage cultures incubated with apoptotic PDAC cells (MØApopCM). MØApopCM pretreatment rendered naïve PDAC cells resistant to Gemcitabine- or Abraxane-induced apoptosis. Proteomic analysis of MØApopCM identified YWHAZ/14-3-3 protein zeta/delta (14-3-3ζ), a major regulator of apoptotic cellular pathways, as a potential mediator of chemoresistance, which was subsequently validated in patient transcriptional datasets, serum samples from PDAC patients and using recombinant 14-3-3ζ and inhibitors thereof. Moreover, in mice bearing orthotopic PDAC tumors, the antitumor potential of Gemcitabine was significantly enhanced by elimination of TAMs using clodronate liposomes or by pharmacological inhibition of the Axl receptor tyrosine kinase, a 14-3-3ζ interacting partner. These data highlight a unique regulatory mechanism by which chemotherapy-induced apoptosis acts as a switch to initiate a protumor/antiapoptotic mechanism in PDAC via 14-3-3ζ/Axl signaling, leading to phosphorylation of Akt and activation of cellular prosurvival mechanisms. The data presented therefore challenge the idea that apoptosis of tumor cells is therapeutically beneficial, at least when immune sensor cells, such as macrophages, are present.

The Ever-Evolving Concept of the Cancer Stem Cell in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer, is the 4th most frequent cause of cancer-related death worldwide, primarily due to the inherent chemoresistant nature and metastatic capacity of this tumor. The latter is believed to be mainly due to the existence of a subpopulation of highly plastic "stem"-like cells within the tumor, known as cancer stem cells (CSCs), which have been shown to have unique metabolic, autophagic, invasive, and chemoresistance properties that allow them to continuously self-renew and escape chemo-therapeutic elimination. As such, current treatments for the majority of PDAC patients are not effective and do not significantly impact overall patient survival (<7 months) as they do not affect the pancreatic CSC (PaCSC) population. In this context, it is important to highlight the need to better understand the characteristics of the PaCSC population in order to develop new therapies to target these cells. In this review, we will provide the latest updates and knowledge on the inherent characteristics of PaCSCs, particularly their unique biological properties including chemoresistance, epithelial to mesenchymal transition, plasticity, metabolism and autophagy.