The Peptidoglycan Recognition Protein 1 confers immune evasive properties on pancreatic cancer stem cells.

OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.

Targeting cancer stem cell OXPHOS with tailored ruthenium complexes as a new anti-cancer strategy.

BACKGROUND: Previous studies by our group have shown that oxidative phosphorylation (OXPHOS) is the main pathway by which pancreatic cancer stem cells (CSCs) meet their energetic requirements; therefore, OXPHOS represents an Achille's heel of these highly tumorigenic cells. Unfortunately, therapies that target OXPHOS in CSCs are lacking. METHODS: The safety and anti-CSC activity of a ruthenium complex featuring bipyridine and terpyridine ligands and one coordination labile position (Ru1) were evaluated across primary pancreatic cancer cultures and in vivo, using 8 patient-derived xenografts (PDXs). RNAseq analysis followed by mitochondria-specific molecular assays were used to determine the mechanism of action. RESULTS: We show that Ru1 is capable of inhibiting CSC OXPHOS function in vitro, and more importantly, it presents excellent anti-cancer activity, with low toxicity, across a large panel of human pancreatic PDXs, as well as in colorectal cancer and osteosarcoma PDXs. Mechanistic studies suggest that this activity stems from Ru1 binding to the D-loop region of the mitochondrial DNA of CSCs, inhibiting OXPHOS complex-associated transcription, leading to reduced mitochondrial oxygen consumption, membrane potential, and ATP production, all of which are necessary for CSCs, which heavily depend on mitochondrial respiration. CONCLUSIONS: Overall, the coordination complex Ru1 represents not only an exciting new anti-cancer agent, but also a molecular tool to dissect the role of OXPHOS in CSCs. Results indicating that the compound is safe, non-toxic and highly effective in vivo are extremely exciting, and have allowed us to uncover unprecedented mechanistic possibilities to fight different cancer types based on targeting CSC OXPHOS.

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.

Bcl3 Couples Cancer Stem Cell Enrichment With Pancreatic Cancer Molecular Subtypes.

BACKGROUND & AIMS: The existence of different subtypes of pancreatic ductal adenocarcinoma (PDAC) and their correlation with patient outcome have shifted the emphasis on patient classification for better decision-making algorithms and personalized therapy. The contribution of mechanisms regulating the cancer stem cell (CSC) population in different subtypes remains unknown. METHODS: Using RNA-seq, we identified B-cell CLL/lymphoma 3 (BCL3), an atypical nf-κb signaling member, as differing in pancreatic CSCs. To determine the biological consequences of BCL3 silencing in vivo and in vitro, we generated bcl3-deficient preclinical mouse models as well as murine cell lines and correlated our findings with human cell lines, PDX models, and 2 independent patient cohorts. We assessed the correlation of bcl3 expression pattern with clinical parameters and subtypes. RESULTS: Bcl3 was significantly down-regulated in human CSCs. Recapitulating this phenotype in preclinical mouse models of PDAC via BCL3 genetic knockout enhanced tumor burden, metastasis, epithelial to mesenchymal transition, and reduced overall survival. Fluorescence-activated cell sorting analyses, together with oxygen consumption, sphere formation, and tumorigenicity assays, all indicated that BCL3 loss resulted in CSC compartment expansion promoting cellular dedifferentiation. Overexpression of BCL3 in human PDXs diminished tumor growth by significantly reducing the CSC population and promoting differentiation. Human PDACs with low BCL3 expression correlated with increased metastasis, and BCL3-negative tumors correlated with lower survival and nonclassical subtypes. CONCLUSIONS: We demonstrate that bcl3 impacts pancreatic carcinogenesis by restraining CSC expansion and by curtailing an aggressive and metastatic tumor burden in PDAC across species. Levels of BCL3 expression are a useful stratification marker for predicting subtype characterization in PDAC, thereby allowing for personalized therapeutic approaches.

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.

Saa3 is a key mediator of the protumorigenic properties of cancer-associated fibroblasts in pancreatic tumors.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by the presence of abundant desmoplastic stroma primarily composed of cancer-associated fibroblasts (CAFs). It is generally accepted that CAFs stimulate tumor progression and might be implicated in drug resistance and immunosuppression. Here, we have compared the transcriptional profile of PDGFRα+ CAFs isolated from genetically engineered mouse PDAC tumors with that of normal pancreatic fibroblasts to identify genes potentially implicated in their protumorigenic properties. We report that the most differentially expressed gene, Saa3, a member of the serum amyloid A (SAA) apolipoprotein family, is a key mediator of the protumorigenic activity of PDGFRα+ CAFs. Whereas Saa3-competent CAFs stimulate the growth of tumor cells in an orthotopic model, Saa3-null CAFs inhibit tumor growth. Saa3 also plays a role in the cross talk between CAFs and tumor cells. Ablation of Saa3 in pancreatic tumor cells makes them insensitive to the inhibitory effect of Saa3-null CAFs. As a consequence, germline ablation of Saa3 does not prevent PDAC development in mice. The protumorigenic activity of Saa3 in CAFs is mediated by Mpp6, a member of the palmitoylated membrane protein subfamily of the peripheral membrane-associated guanylate kinases (MAGUK). Finally, we interrogated whether these observations could be translated to a human scenario. Indeed, SAA1, the ortholog of murine Saa3, is overexpressed in human CAFs. Moreover, high levels of SAA1 in the stromal component correlate with worse survival. These findings support the concept that selective inhibition of SAA1 in CAFs may provide potential therapeutic benefit to PDAC patients.

The miR-25-93-106b cluster regulates tumor metastasis and immune evasion via modulation of CXCL12 and PD-L1.

The stromal microenvironment controls response to injury and inflammation, and is also an important determinant of cancer cell behavior. However, our understanding of its modulation by miRNA (miR) and their respective targets is still sparse. Here, we identified the miR-25-93-106b cluster and two new target genes as critical drivers for metastasis and immune evasion of cancer cells. Using miR-25-93-106b knockout mice or antagomiRs, we demonstrated regulation of the production of the chemoattractant CXCL12 controlling bone marrow metastasis. Moreover, we identified the immune checkpoint PD-L1 (CD274) as a novel miR-93/106b target playing a central role in diminishing tumor immunity. Eventually, upregulation of miR-93 and miR-106b via miR-mimics or treatment with an epigenetic reader domain (BET) inhibitor resulted in diminished expression of CXCL12 and PD-L1. These data suggest a potential new therapeutic rationale for use of BET inhibitors for dual targeting of cancers with strong immunosuppressive and metastatic phenotypes.

Cancer Stem Cells and Macrophages: Implications in Tumor Biology and Therapeutic Strategies.

Cancer stem cells (CSCs) are a unique subset of cells within tumors with stemlike properties that have been proposed to be key drivers of tumor initiation and progression. CSCs are functionally defined by their unlimited self-renewal capacity and their ability to initiate tumor formation in vivo. Like normal stem cells, CSCs exist in a cellular niche comprised of numerous cell types including tumor-associated macrophages (TAMs) which provides a unique microenvironment to protect and promote CSC functions. TAMs provide pivotal signals to promote CSC survival, self-renewal, maintenance, and migratory ability, and in turn, CSCs deliver tumor-promoting cues to TAMs that further enhance tumorigenesis. Studies in the last decade have aimed to understand the molecular mediators of CSCs and TAMs, and recent advances have begun to elucidate the complex cross talk that occurs between these two cell types. In this review, we discuss the molecular interactions that define CSC-TAM cross talk at each stage of tumor progression and examine the clinical implications of targeting these interactions.

B Lymphocyte commitment program is driven by the proto-oncogene c-Myc.

c-Myc, a member of the Myc family of transcription factors, is involved in numerous biological functions including the regulation of cell proliferation, differentiation, and apoptosis in various cell types. Of all of its functions, the role of c-Myc in cell differentiation is one of the least understood. We addressed the role of c-Myc in B lymphocyte differentiation. We found that c-Myc is essential from early stages of B lymphocyte differentiation in vivo and regulates this process by providing B cell identity via direct transcriptional regulation of the ebf-1 gene. Our data show that c-Myc influences early B lymphocyte differentiation by promoting activation of B cell identity genes, thus linking this transcription factor to the EBF-1/Pax-5 pathway.

c-Myc regulates cell size and ploidy but is not essential for postnatal proliferation in liver.

The c-Myc protein is a transcription factor implicated in the regulation of multiple biological processes, including cell proliferation, cell growth, and apoptosis. In vivo overexpression of c-myc is linked to tumor development in a number of mouse models. Here, we show that perinatal inactivation of c-Myc in liver causes disorganized organ architecture, decreased hepatocyte size, and cell ploidy. Furthermore, c-Myc appears to have distinct roles in proliferation in liver. Thus, postnatal hepatocyte proliferation does not require c-Myc, whereas it is necessary for liver regeneration in adult mice. These results show novel physiological functions of c-myc in liver development and hepatocyte proliferation and growth.