ZEB1 shapes AML immunological niches, suppressing CD8 T cell activity while fostering Th17 cell expansion.

Acute myeloid leukemia (AML) progression is influenced by immune suppression induced by leukemia cells. ZEB1, a critical transcription factor in epithelial-to-mesenchymal transition, demonstrates immune regulatory functions in AML. Silencing ZEB1 in leukemic cells reduces engraftment and extramedullary disease in immune-competent mice, activating CD8 T lymphocytes and limiting Th17 cell expansion. ZEB1 in AML cells directly promotes Th17 cell development that, in turn, creates a self-sustaining loop and a pro-invasive phenotype, favoring transforming growth factor ß (TGF-ß), interleukin-23 (IL-23), and SOCS2 gene transcription. In bone marrow biopsies from AML patients, immunohistochemistry shows a direct correlation between ZEB1 and Th17. Also, the analysis of ZEB1 expression in larger datasets identifies two distinct AML groups, ZEB1high and ZEB1low, each with specific immunological and molecular traits. ZEB1high patients exhibit increased IL-17, SOCS2, and TGF-ß pathways and a negative association with overall survival. This unveils ZEB1's dual role in AML, entwining pro-tumoral and immune regulatory capacities in AML blasts.

JMJD6 Shapes a Pro-tumor Microenvironment via ANXA1-Dependent Macrophage Polarization in Breast Cancer.

Breast cancer is the most common type of cancer in women worldwide, with the luminal subtype being the most widespread. Although characterized by better prognosis compared with other subtypes, luminal breast cancer is still considered a threatening disease due to therapy resistance, which occurs via both cell- and non-cell-autonomous mechanisms. Jumonji domain-containing 6, arginine demethylase and lysine hydroxylase (JMJD6) is endowed with a negative prognostic value in luminal breast cancer and, via its epigenetic activity, it is known to regulate many intrinsic cancer cell pathways. So far, the effect of JMJD6 in molding the surrounding microenvironment has not been explored.Here, we describe a novel function of JMJD6 showing that its genetic inhibition in breast cancer cells suppresses lipid droplet formation and ANXA1 expression, via estrogen receptor alpha and PPARα modulation. Reduction of intracellular ANXA1 results in decreased release in the tumor microenvironment (TME), ultimately preventing M2-type macrophage polarization and tumor aggressiveness. IMPLICATIONS: Our findings identify JMJD6 as a determinant of breast cancer aggressiveness and provide the rationale for the development of inhibitory molecules to reduce disease progression also through the remodeling of TME composition.

ATF3 Reprograms the Bone Marrow Niche in Response to Early Breast Cancer Transformation.

Cancer is a systemic disease able to reprogram the bone marrow (BM) niche towards a protumorigenic state. The impact of cancer on specific BM subpopulations can qualitatively differ according to the signals released by the tumor, which can vary on the basis of the tissue of origin. Using a spontaneous model of mammary carcinoma, we identified BM mesenchymal stem cells (MSC) as the first sensors of distal cancer cells and key mediators of BM reprogramming. Through the release of IL1B, BM MSCs induced transcriptional upregulation and nuclear translocation of the activating transcription factor 3 (ATF3) in hematopoietic stem cells. ATF3 in turn promoted the formation of myeloid progenitor clusters and sustained myeloid cell differentiation. Deletion of Atf3 specifically in the myeloid compartment reduced circulating monocytes and blocked their differentiation into tumor-associated macrophages. In the peripheral blood, the association of ATF3 expression in CD14+ mononuclear cells with the expansion CD11b+ population was able to discriminate between women with malignant or benign conditions at early diagnosis. Overall, this study identifies the IL1B/ATF3 signaling pathway in the BM as a functional step toward the establishment of a tumor-promoting emergency myelopoiesis, suggesting that ATF3 could be tested in a clinical setting as a circulating marker of early transformation and offering the rationale for testing the therapeutic benefits of IL1B inhibition in patients with breast cancer. Significance: Bone marrow mesenchymal stem cells respond to early breast tumorigenesis by upregulating IL1B to promote ATF3 expression in hematopoietic stem cells and to induce myeloid cell differentiation that supports tumor development.

Gene signatures of circulating breast cancer cell models are a source of novel molecular determinants of metastasis and improve circulating tumor cell detection in patients.

BACKGROUND: Progression to stage IV disease remains the main cause of breast cancer-related deaths. Increasing knowledge on the hematogenous phase of metastasis is key for exploiting the entire window of opportunity to interfere with early dissemination and to achieve a more effective disease control. Recent evidence suggests that circulating tumor cells (CTCs) possess diverse adaptive mechanisms to survive in blood and eventually metastasize, encouraging research into CTC-directed therapies. METHODS: On the hypothesis that the distinguishing molecular features of CTCs reveal useful information on metastasis biology and disease outcome, we compared the transcriptome of CTCs, primary tumors, lymph-node and lung metastases of the MDA-MB-231 xenograft model, and assessed the biological role of a panel of selected genes, by in vitro and in vivo functional assays, and their clinical significance in M0 and M+ breast cancer patients. RESULTS: We found that hematogenous dissemination is governed by a transcriptional program and identified a CTC signature that includes 192 up-regulated genes, mainly related to cell plasticity and adaptation, and 282 down-regulated genes, involved in chromatin remodeling and transcription. Among genes up-regulated in CTCs, FADS3 was found to increases cell membrane fluidity and promote hematogenous diffusion and lung metastasis formation. TFF3 was observed to be associated with a subset of CTCs with epithelial-like features in the experimental model and in a cohort of 44 breast cancer patients, and to play a role in cell migration, invasion and blood-borne dissemination. The analysis of clinical samples with a panel of CTC-specific genes (ADPRHL1, ELF3, FCF1, TFF1 and TFF3) considerably improved CTC detection as compared with epithelial and tumor-associated markers both in M0 and stage IV patients, and CTC kinetics informed disease relapse in the neoadjuvant setting. CONCLUSIONS: Our findings provide evidence on the potential of a CTC-specific molecular profile as source of metastasis-relevant genes in breast cancer experimental models and in patients. Thanks to transcriptome analysis we generated a novel CTC signature in the MDA-MB-231 xenograft model, adding a new piece to the current knowledge on the key players that orchestrate tumor cell hematogenous dissemination and breast cancer metastasis, and expanding the list of CTC-related biomarkers for future validation studies.

Few, but Efficient: The Role of Mast Cells in Breast Cancer and Other Solid Tumors.

Tumor outcome is determined not only by cancer cell-intrinsic features but also by the interaction between cancer cells and their microenvironment. There is great interest in tumor-infiltrating immune cells, yet mast cells have been less studied. Recent work has highlighted the impact of mast cells on the features and aggressiveness of cancer cells, but the eventual effect of mast cell infiltration is still controversial. Here, we review multifaceted findings regarding the role of mast cells in cancer, with a particular focus on breast cancer, which is further complicated because of its classification into subtypes characterized by different biological features, outcome, and therapeutic strategies.

Structure-based identification of a new IAP-targeting compound that induces cancer cell death inducing NF-κB pathway.

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein-protein interaction surfaces, can affect events occurring at different stages of cancer development. To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

Deciphering the nonsense-mediated mRNA decay pathway to identify cancer cell vulnerabilities for effective cancer therapy.

Nonsense-mediated mRNA decay (NMD) is a highly conserved cellular surveillance mechanism, commonly studied for its role in mRNA quality control because of its capacity of degrading mutated mRNAs that would produce truncated proteins. However, recent studies have proven that NMD hides more complex tasks involved in a plethora of cellular activities. Indeed, it can control the stability of mutated as well as non-mutated transcripts, tuning transcriptome regulation. NMD not only displays a pivotal role in cell physiology but also in a number of genetic diseases. In cancer, the activity of this pathway is extremely complex and it is endowed with both pro-tumor and tumor suppressor functions, likely depending on the genetic context and tumor microenvironment. NMD inhibition has been tested in pre-clinical studies showing favored production of neoantigens by cancer cells, which can stimulate the triggering of an anti-tumor immune response. At the same time, NMD inhibition could result in a pro-tumor effect, increasing cancer cell adaptation to stress. Since several NMD inhibitors are already available in the clinic to treat genetic diseases, these compounds could be redirected to treat cancer patients, pending the comprehension of these variegated NMD regulation mechanisms. Ideally, an effective strategy should exploit the anti-tumor advantages of NMD inhibition and simultaneously preserve its intrinsic tumor suppressor functions. The targeting of NMD could provide a new therapeutic opportunity, increasing the immunogenicity of tumors and potentially boosting the efficacy of the immunotherapy agents now available for cancer treatment.

SPARC regulation of PMN clearance protects from pristane-induced lupus and rheumatoid arthritis.

The secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein with unexpected immunosuppressive function in myeloid cells. We investigated the role of SPARC in autoimmunity using the pristane-induced model of lupus that, in mice, mimics human systemic lupus erythematosus (SLE). Sparc -/- mice developed earlier and more severe renal disease, multi-organ parenchymal damage, and arthritis than the wild-type counterpart. Sparc +/- heterozygous mice showed an intermediate phenotype suggesting Sparc gene dosage in autoimmune-related events. Mechanistically, reduced Sparc expression in neutrophils blocks their clearance by macrophages, through defective delivery of don't-eat-me signals. Dying Sparc -/- neutrophils that escape macrophage scavenging become source of autoantigens for dendritic cell presentation and are a direct stimulation for γδT cells. Gene profile analysis of knee synovial biopsies from SLE-associated arthritis showed an inverse correlation between SPARC and key autoimmune genes. These results point to SPARC down-regulation as a leading event characterizing SLE and rheumatoid arthritis pathogenesis.

T Cells Expressing Receptor Recombination/Revision Machinery Are Detected in the Tumor Microenvironment and Expanded in Genomically Over-unstable Models.

Tumors undergo dynamic immunoediting as part of a process that balances immunologic sensing of emerging neoantigens and evasion from immune responses. Tumor-infiltrating lymphocytes (TIL) comprise heterogeneous subsets of peripheral T cells characterized by diverse functional differentiation states and dependence on T-cell receptor (TCR) specificity gained through recombination events during their development. We hypothesized that within the tumor microenvironment (TME), an antigenic milieu and immunologic interface, tumor-infiltrating peripheral T cells could reexpress key elements of the TCR recombination machinery, namely, Rag1 and Rag2 recombinases and Tdt polymerase, as a potential mechanism involved in the revision of TCR specificity. Using two syngeneic invasive breast cancer transplantable models, 4T1 and TS/A, we observed that Rag1, Rag2, and Dntt in situ mRNA expression characterized rare tumor-infiltrating T cells. In situ expression of the transcripts was increased in coisogenic Mlh1-deficient tumors, characterized by genomic overinstability, and was also modulated by PD-1 immune-checkpoint blockade. Through immunolocalization and mRNA hybridization analyses, we detected the presence of rare TDT+RAG1/2+ cells populating primary tumors and draining lymph nodes in human invasive breast cancer. Analysis of harmonized single-cell RNA-sequencing data sets of human cancers identified a very small fraction of tumor-associated T cells, characterized by the expression of recombination/revision machinery transcripts, which on pseudotemporal ordering corresponded to differentiated effector T cells. We offer thought-provoking evidence of a TIL microniche marked by rare transcripts involved in TCR shaping.

Immune-tolerance to human iPS-derived neural progenitors xenografted into the immature cerebellum is overridden by species-specific differences in differentiation timing.

We xeno-transplanted human neural precursor cells derived from induced pluripotent stem cells into the cerebellum and brainstem of mice and rats during prenatal development or the first postnatal week. The transplants survived and started to differentiate up to 1 month after birth when they were rejected by both species. Extended survival and differentiation of the same cells were obtained only when they were transplanted in NOD-SCID mice. Transplants of human neural precursor cells mixed with the same cells after partial in vitro differentiation or with a cellular extract obtained from adult rat cerebellum increased survival of the xeno-graft beyond one month. These findings are consistent with the hypothesis that the slower pace of differentiation of human neural precursors compared to that of rodents restricts induction of immune-tolerance to human antigens expressed before completion of maturation of the immune system. With further maturation the transplanted neural precursors expressed more mature antigens before the graft were rejected. Supplementation of the immature cells suspensions with more mature antigens may help to induce immune-tolerance for those antigens expressed only later by the engrafted cells.

Infiltrating Mast Cell-Mediated Stimulation of Estrogen Receptor Activity in Breast Cancer Cells Promotes the Luminal Phenotype.

Tumor growth and development is determined by both cancer cell-autonomous and microenvironmental mechanisms, including the contribution of infiltrating immune cells. Because the role of mast cells (MC) in this process is poorly characterized and even controversial, we investigated their part in breast cancer. Crossing C57BL/6 MMTV-PyMT mice, which spontaneously develop mammary carcinomas, with MC-deficient C57BL/6-KitW-sh/W-sh (Wsh) mice, showed that MCs promote tumor growth and prevent the development of basal CK5-positive areas in favor of a luminal gene program. When cocultured with breast cancer cells in vitro, MCs hindered activation of cMET, a master regulator of the basal program, and simultaneously promoted expression and activation of estrogen receptor (ESR1/ER) and its target genes (PGR, KRT8/CK8, BCL2), which are all luminal markers. Moreover, MCs reduced ERBB2/HER2 levels, whose inhibition further increased ESR1 expression. In vivo and in silico analysis of patients with breast cancer revealed a direct correlation between MC density and ESR1 expression. In mice engrafted with HER2-positive breast cancer tumors, coinjection of MCs increased tumor engraftment and outgrowth, supporting the link between MCs and increased risk of relapse in patients with breast cancer. Together, our findings support the notion that MCs influence the phenotype of breast cancer cells by stimulating a luminal phenotype and ultimately modifying the outcome of the disease. SIGNIFICANCE: Mast cells impact breast cancer outcome by directly affecting the phenotype of tumor cells through stimulation of the estrogen receptor pathway.

Immune Checkpoint Ligand Reverse Signaling: Looking Back to Go Forward in Cancer Therapy.

The so-called immune checkpoints are pathways that regulate the timing and intensity of the immune response to avoid an excessive reaction and to protect the host from autoimmunity. Immune checkpoint inhibitors (ICIs) are designed to target the negative regulatory pathways of T cells, and they have been shown to restore anti-tumor immune functions and achieve considerable clinical results. Indeed, several clinical trials have reported durable clinical response in different tumor types, such as melanoma, renal cell carcinoma (RCC) and non-small cell lung cancer (NSCLC). Nonetheless, after the initial enthusiasm, it is now evident that the majority of patients do not benefit from ICIs, due to innate or acquired tumor resistance. It is therefore mandatory to find ways to identify those patients who will respond and to find ways to induce response in those who at present do not benefit from ICIs. In this regard, the expression of programmed death ligand 1 (PD-L1) on neoplastic cells was the first, and most obvious, biomarker exploited to predict the activity of anti-programmed death 1 (PD-1) and/or anti-PD-L1 antibodies. As expected, a correlation was confirmed between the levels of PD-L1 and the efficacy of anti-PD-1 therapy in melanoma, NSCLC and RCC. However, further results from clinical trials showed that some patients display a clinical response regardless of tumor cell PD-L1 expression levels, while others do not benefit from ICI treatment despite the expression of PD-L1 on neoplastic elements. These findings strongly support the notion that other factors may be relevant for the efficacy of ICI-based treatment regimens. Furthermore, although the current dogma indicates that the PD-1/PD-L1 axis exerts its regulatory effects via the signal transduced in PD-1-expressing T cells, recent evidence suggests that a reverse signaling may also exist downstream of PD-L1 in both tumor and immune cells. The reverse signaling of PD-L1, but also of other immune checkpoints, might contribute to the pro-tumoral/immune suppressive environment associated with tumor development and progression. Clarifying this aspect could facilitate the prediction of patients' clinical outcomes, which are so far unpredictable and result in response, resistance or even hyper-progressive disease in some cases.

When Failure Is Worse Than Giving Up: The Case of CTL.

Although much emphasis is given to the use of immune checkpoint inhibitors to restore the functionality of exhausted lymphocytes, very little is known about the fate of cancer cells that escape from the cytotoxic activity of T cells. In a previous issue of Cancer Research, Stein and colleagues investigated the response of cancer cells to CD8+ T cells disarmed of their killing activity. Spared cancer cells acquired stem cell-like features and displayed an enhanced capacity to form tumors and metastasize. These increased tumorigenic properties could represent the other side of the coin of T-cell surveillance seen in wound healing in which recognition of damaged tissue as "self" gives the green light for healing process.See related article by Stein and colleagues; Cancer Res 79(7):1507-19.

Targeting the BIR Domains of Inhibitor of Apoptosis (IAP) Proteins in Cancer Treatment.

Inhibitor of apoptosis (IAP) proteins are characterized by the presence of the conserved baculoviral IAP repeat (BIR) domain that is involved in protein-protein interactions. IAPs were initially thought to be mainly responsible for caspase inhibition, acting as negative regulators of apoptosis, but later works have shown that IAPs also control a plethora of other different cellular pathways. As X-linked IAP (XIAP), and other IAP, levels are often deregulated in cancer cells and have been shown to correlate with patients' prognosis, several approaches have been pursued to inhibit their activity in order to restore apoptosis. Many small molecules have been designed to target the BIR domains, the vast majority being inspired by the N-terminal tetrapeptide of Second Mitochondria-derived Activator of Caspases/Direct IAp Binding with Low pI (Smac/Diablo), which is the natural XIAP antagonist. These compounds are therefore usually referred to as Smac mimetics (SMs). Despite the fact that SMs were intended to specifically target XIAP, it has been shown that they also interact with cellular IAP-1 (cIAP1) and cIAP2, promoting their proteasome-dependent degradation. SMs have been tested in combination with several cytotoxic compounds and are now considered promising immune modulators which can be exploited in cancer therapy, especially in combination with immune checkpoint inhibitors. In this review, we give an overview of the structural hot-spots of BIRs, focusing on their fold and on the peculiar structural patches which characterize the diverse BIRs. These structures are exploited/exploitable for the development of specific and active IAP inhibitors.

DNA damage and transcriptional regulation in iPSC-derived neurons from Ataxia Telangiectasia patients.

Ataxia Telangiectasia (A-T) is neurodegenerative syndrome caused by inherited mutations inactivating the ATM kinase, a master regulator of the DNA damage response (DDR). What makes neurons vulnerable to ATM loss remains unclear. In this study we assessed on human iPSC-derived neurons whether the abnormal accumulation of DNA-Topoisomerase 1 adducts (Top1ccs) found in A-T impairs transcription elongation, thus favoring neurodegeneration. Furthermore, whether neuronal activity-induced immediate early genes (IEGs), a process involving the formation of DNA breaks, is affected by ATM deficiency. We found that Top1cc trapping by CPT induces an ATM-dependent DDR as well as an ATM-independent induction of IEGs and repression especially of long genes. As revealed by nascent RNA sequencing, transcriptional elongation and recovery were found to proceed with the same rate, irrespective of gene length and ATM status. Neuronal activity induced by glutamate receptors stimulation, or membrane depolarization with KCl, triggered a DDR and expression of IEGs, the latter independent of ATM. In unperturbed A-T neurons a set of genes (FN1, DCN, RASGRF1, FZD1, EOMES, SHH, NR2E1) implicated in the development, maintenance and physiology of central nervous system was specifically downregulated, underscoring their potential involvement in the neurodegenerative process in A-T patients.

Mitosis perturbation by MASTL depletion impairs the viability of thyroid tumor cells.

Even if thyroid tumors are generally curable, a fraction will develop resistance to therapy and progress towards undifferentiated forms, whose treatment remains a demanding challenge. To identify potential novel targets for treatment of thyroid cancer, in a previous study using siRNA-mediated functional screening, we identified several genes that are essential for the growth of thyroid tumor, but not normal cells. Among the top-ranking hits, we found microtubule associated serine/threonine kinase-like (MASTL), which is known to play an essential role in mitosis regulation, and is also involved in the DNA damage response. Herein, we examine the effects of MASTL depletion on growth and viability of thyroid tumor cells. MASTL depletion impaired cell proliferation and increased the percentage of cells presenting nuclear anomalies, which are indicative of mitotic catastrophe. Furthermore, MASTL depletion was associated with enhanced DNA damage. All these effects eventually led to cell death, characterized by the presence of apoptotic markers. Moreover, MASTL depletion sensitized thyroid tumor cells to cisplatin. Our results demonstrate that MASTL represents vulnerability for thyroid tumor cells, which could be explored as a therapeutic target for thyroid cancer.

Structure-based design and molecular profiling of Smac-mimetics selective for cellular IAPs.

Inhibitor of Apoptosis Proteins (IAPs) is highly conserved negative regulators of apoptosis overexpressed in many cancer cells. Based on their endogenous antagonist, Smac/DIABLO, mimic compounds (Smac-mimetics, SMs) have been developed to inhibit IAPs prosurvival activity, showing promising effects in advanced phases of clinical trials. Since different IAP homologs play distinctive roles in cancer cell survival and immunomodulation, SM-induced apoptosis proceeds through diverse mechanisms. After binding to their BIR3 domain, SMs have been shown to rapidly induce auto-ubiquitylation and degradation of cellular IAPs (cIAPs), thus leading to cell death mainly by activation of the noncanonical NF-κB pathway. For this reason, we started the BIR3-driven design of compounds selective for cIAP1 and with reduced affinity for X-linked IAP (XIAP), in order to focus SMs antitumor activity on cIAPs degradation. In this work, we describe the crystal structures of the BIR3 domains of cIAP1 and XIAP, each in complex with a cIAP1-selective SM (SM130 and SM114, respectively). The two SMs displayed 23- and 32-fold higher affinity for cIAP1-BIR3 over XIAP-BIR3 in molecular displacement experiments based on fluorescence polarization. In vitro cell-based assays confirmed that both selective SMs triggered apoptosis in cancer cells with different efficiencies by inducing caspases-3, -8, and -9-independent cIAP1 degradation. The design of cIAPs-selective compounds represents an innovative approach in the field of anticancer drugs development, being useful to elucidate different prosurvival mechanisms and to reduce the adverse effects of pan-IAPs compounds in cancer therapy. DATABASE: Structural data are available in the Protein Data Bank database under the accession codes 6EXW (cIAP1-BIR3/SM130 complex) and 6EY2 (XIAP-BIR3/SM114 complex).

cIAP1 regulates the EGFR/Snai2 axis in triple-negative breast cancer cells.

Inhibitor of apoptosis (IAP) proteins constitute a family of conserved molecules that regulate both apoptosis and receptor signaling. They are often deregulated in cancer cells and represent potential targets for therapy. In our work, we investigated the effect of IAP inhibition in vivo to identify novel downstream genes expressed in an IAP-dependent manner that could contribute to cancer aggressiveness. To this end, immunocompromised mice engrafted subcutaneously with the triple-negative breast cancer MDA-MB231 cell line were treated with SM83, a Smac mimetic that acts as a pan-IAP inhibitor, and tumor nodules were profiled for gene expression. SM83 reduced the expression of Snai2, an epithelial-to-mesenchymal transition factor often associated with increased stem-like properties and metastatic potential especially in breast cancer cells. By testing several breast cancer cell lines, we demonstrated that Snai2 downregulation prevents cell motility and that its expression is promoted by cIAP1. In fact, the chemical or genetic inhibition of cIAP1 blocked epidermal growth factor receptor (EGFR)-dependent activation of the mitogen-activated protein kinase (MAPK) pathway and caused the reduction of Snai2 transcription levels. In a number of breast cancer cell lines, cIAP1 depletion also resulted in a reduction of EGFR protein levels which derived from the decrease of its gene transcription, though, paradoxically, the silencing of cIAP1 promoted EGFR protein stability rather than its degradation. Finally, we provided evidence that IAP inhibition displays an anti-tumor and anti-metastasis effect in vivo. In conclusion, our work indicates that IAP-targeted therapy could contribute to EGFR inhibition and to the reduction of its downstream mediators. This approach could be particularly effective in tumors characterized by high levels of EGFR and Snai2, such as triple-negative breast cancer.

MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner.

BACKGROUND: Fibroblasts are crucial mediators of tumor-stroma cross-talk through synthesis and remodeling of the extracellular matrix and production of multiple soluble factors. Nonetheless, little is still known about specific determinants of fibroblast pro-tumorigenic activity in lung cancer. Here, we aimed at understanding the role of miRNAs, which are often altered in stromal cells, in reprogramming fibroblasts towards a tumor-supporting phenotype. METHODS: We employed a co-culture-based high-throughput screening to identify specific miRNAs modulating the pro-tumorigenic potential of lung fibroblasts. Multiplex assays and ELISA were instrumental to study the effect of miRNAs on the secretome of both primary and immortalized lung fibroblasts from lung cancer patients and to evaluate plasmatic levels of HGF in heavy smokers. Direct mRNA targeting by miRNAs was investigated through dual-luciferase reporter assay and western blot. Finally, the pro-tumorigenic activity of fibroblasts and their conditioned media was tested by employing in vitro migration experiments and mouse xenografts. RESULTS: We identified miR-16 as a master regulator of fibroblast secretome and showed that its upregulation reduces HGF secretion by fibroblasts, impairing their capacity to promote cancer cell migration. This effect is due to a pleiotropic activity of miR-16 which prevents HGF expression through direct inhibition of FGFR-1 signaling and targeting of HGF mRNA. Mechanistically, miR-16 targets FGFR-1 downstream mediator MEK1, thus reducing ERK1/2 activation. Consistently, chemical or genetic inhibition of FGFR-1 mimics miR-16 activity and prevents HGF production. Of note, we report that primary fibroblast cell lines derived from lungs of heavy smokers express reduced miR-16 levels compared to those from lungs not exposed to smoke and that HGF concentration in heavy smokers' plasma correlates with levels of tobacco exposure. Finally, in vivo experiments confirmed that restoration of miR-16 expression in fibroblasts reduced their ability to promote tumor growth and that HGF plays a central role in the pro-tumorigenic activity of fibroblasts. CONCLUSIONS: Overall, these results uncover a central role for miR-16 in regulating HGF production by lung fibroblasts, thus affecting their pro-tumorigenic potential. Correlation between smoking exposure and miR-16 levels could provide novel clues regarding the formation of a tumor-proficient milieu during the early phases of lung cancer development.

Supersulfated low-molecular weight heparin synergizes with IGF1R/IR inhibitor to suppress synovial sarcoma growth and metastases.

Synovial sarcoma (SS) is an aggressive tumor with propensity for lung metastases which significantly impact patients' prognosis. New therapeutic approaches are needed to improve treatment outcome. Targeting the heparanase/heparan sulfate proteoglycan system by heparin derivatives which act as heparanase inhibitors/heparan sulfate mimetics is emerging as a therapeutic approach that can sensitize the tumor response to chemotherapy. We investigated the therapeutic potential of a supersulfated low molecular weight heparin (ssLMWH) in preclinical models of SS. ssLMWH showed a potent anti-heparanase activity, dose-dependently inhibited SS colony growth and cell invasion, and downregulated the activation of receptor tyrosine kinases including IGF1R and IR. The combination of ssLMWH and the IGF1R/IR inhibitor BMS754807 synergistically inhibited proliferation of cells exhibiting IGF1R hyperactivation, also abrogating cell motility and promoting apoptosis in association with PI3K/AKT pathway inhibition. The drug combination strongly enhanced the antitumor effect against the CME-1 model, as compared to single agent treatment, abrogating orthotopic tumor growth and significantly repressing spontaneous lung metastatic dissemination in treated mice. These findings provide a strong preclinical rationale for developing drug regimens combining heparanase inhibitors/HS mimetics with IGF1R antagonists for treatment of metastatic SS.