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Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse.Significance:CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival.This article is highlighted in the In This Issue feature, p. 1171

Supplementary Figure from Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer

Supplementary Data from Tumor ENPP1 (CD203a)/Haptoglobin Axis Exploits Myeloid-Derived Suppressor Cells to Promote Post-Radiotherapy Local Recurrence in Breast Cancer

Supplementary Methods: For the characterization of the L-aptamer AON-D21 Supplementary Results: Characterization of the L-aptamer AON-D21 Supplementary References Supplementary Figure S1: Pharmacokinetics of AON-D21 Supplementary Figure S2: Individual follow up of subcutaneous growth and rechallenge Supplementary Figure S3: Splenic leukocyte subpopulations in mice bearing 393P tumors Supplementary Figure S4: Scatter plots of CD8 T cells in treated 393P tumors Supplementary Figure S5: Tumor-infiltrating leukocyte subpopulations in 393P tumors Supplementary Figure S6: Splenic leukocyte subpopulations in mice bearing LLC tumors Supplementary Figure S7: Tumor-infiltrating leukocyte subpopulations and IL2 in LLC tumors Supplementary Figure S8: Flow cytometry gating strategy Supplementary Table S1: Rate constants and dissociation constant of AON-D21 Supplementary Table S2: mRNA expression of immune-related molecules in 393P tumors

An Id1 gene signature is dependent of KEAP1-related genes and a FOSL1 network.

Clinical implication of Id1 expression in LUAD patients.

Supplementary tables and figure's headings

Activating mutations in some isoforms of RAS or RAF are drivers of a substantial proportion of cancers. The main Raf effector, MEK1/2, can be targeted with several highly specific inhibitors. The clinical activity of these inhibitors seems to be mixed, showing efficacy against mutant BRAF-driven tumors but not KRAS-driven tumors, such as pancreatic adenocarcinomas. To improve our understanding of this context-dependent efficacy, we generated pancreatic cancer cells resistant to MEK1/2 inhibition, which were also resistant to KRAS and ERK1/2 inhibitors. Compared with parental cells, inhibitor-resistant cells showed several phenotypic changes including increased metastatic ability in vivo. The transcription factor SLUG, which is known to induce epithelial-to-mesenchymal transition, was identified as the key factor responsible for both resistance to MEK1/2 inhibition and increased metastasis. Slug, but not similar transcription factors, predicted poor prognosis of pancreatic cancer patients and induced the transition to a cellular phenotype in which cell-cycle progression becomes independent of the KRAS–RAF–MEK1/2–ERK1/2 pathway. SLUG was targeted using two independent strategies: (i) inhibition of the MEK5–ERK5 pathway, which is responsible for upregulation of SLUG upon MEK1/2 inhibition, and (ii) direct PROTAC-mediated degradation. Both strategies were efficacious in preclinical pancreatic cancer models, paving the path for the development of more effective therapies against pancreatic cancer.Significance:This study demonstrates that SLUG confers resistance to MEK1/2 inhibitors in pancreatic cancer by uncoupling tumor progression from KRAS–RAF–MEK1/2–ERK1/2 signaling, providing new therapeutic opportunities.

Because of the refractory nature of mutant KRAS lung adenocarcinoma (LUAD) to current therapies, identification of new molecular targets is essential. Genes with a prognostic role in mutant KRAS LUAD have proven to be potential molecular targets for therapeutic development. Here we determine the clinical, functional, and mechanistic role of inhibitor of differentiation-1 (Id1) in mutant KRAS LUAD. Analysis of LUAD cohorts from TCGA and SPORE showed that high expression of Id1 was a marker of poor survival in patients harboring mutant, but not wild-type KRAS. Abrogation of Id1 induced G2–M arrest and apoptosis in mutant KRAS LUAD cells. In vivo, loss of Id1 strongly impaired tumor growth and maintenance as well as liver metastasis, resulting in improved survival. Mechanistically, Id1 was regulated by the KRAS oncogene through JNK, and loss of Id1 resulted in downregulation of elements of the mitotic machinery via inhibition of the transcription factor FOSL1 and of several kinases within the KRAS signaling network. Our study provides clinical, functional, and mechanistic evidence underscoring Id1 as a critical gene in mutant KRAS LUAD and warrants further studies of Id1 as a therapeutic target in patients with LUAD.Significance:These findings highlight the prognostic significance of the transcriptional regulator Id1 in KRAS-mutant lung adenocarcinoma and provide mechanistic insight into how it controls tumor growth and metastasis.

A shRNA-resistant Id1 cDNA rescues Id1 loss.

Extended Material and Methods

Regulation of kinases by Id1 expression

PDF file - 462KB, Forest plots summarizing the overexpression of the 11 genes from discovery meta-analysis (S1). Forest plots summarizing the overexpression of the 11 genes from validation meta-analysis (S2). Six genes of the original 11 gene ADC signature are also overexpressed in ADC relative to other NSCLC histological subtypes (S3). Expression of PTK7 in an ADC tissue microarray and in a mouse model of lung adenocarcinoma (S4). Correlation of sensitivity to PTK7 knock-down with mutations and expression (S5). MKK7-JNK pathway dysregulation upon PTK7 knock-down (S6).

XLS file - 27K, Onto-Express Analysis for Functional Pathways

PDF file - 20K, Survival prognosis of a mouse CAF-secretory gene signature in human NSCLC

PDF file - 40K, Lung CAFs stimulate lung cancer growth in vivo

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XLS file - 329K, Microarray Analysis using LIMMA

Bone is a frequent target of lung cancer metastasis, which is associated with significant morbidity and a dismal prognosis. To identify and functionally characterize genes involved in the mechanisms of osseous metastasis, we developed a murine lung cancer model. Comparative transcriptomic analysis identified genes encoding signaling molecules (such as TCF4 and PRKD3) and cell anchorage–related proteins (MCAM and SUSD5), some of which were basally modulated by transforming growth factor-β (TGF-β) in tumor cells and in conditions mimicking tumor-stromal interactions. Triple gene combinations induced not only high osteoclastogenic activity but also a marked enhancement of global metalloproteolytic activities in vitro. These effects were strongly associated with robust bone colonization in vivo, whereas this gene subset was ineffective in promoting local tumor growth and cell homing activity to bone. Interestingly, global inhibition of metalloproteolytic activities and simultaneous TGF-β blockade in vivo led to increased survival and a remarkable attenuation of bone tumor burden and osteolytic metastasis. Thus, this metastatic gene signature mediates bone matrix degradation by a dual mechanism of induction of TGF-β–dependent osteoclastogenic bone resorption and enhancement of stroma-dependent metalloproteolytic activities. Our findings suggest the cooperative contribution of host-derived and cell autonomous effects directed by a small subset of genes in mediating aggressive osseous colonization. [Cancer Res 2008;68(7):2275–85]

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Supplementary Figure Legends 1-7 from A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism

XLS file - 21K, Ingenuity Pathway Analysis for Functional Networks

XLS file - 27K, Meta-Analysis of 5 human data sets incorporating array expression data

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Supplementary Table A, Figures 1-7 from A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism

PDF file - 56K, A secretory and inflammatory gene signature specific to CAFs

PDF file - 85K, Lung tumor cells and CAFs induce a of CAF-like phenotype in NFs

XLSX file - 40KB, Data sets used in discovery meta-analysis (S1). Data sets used in validation meta-analysis (S2). Gene-level results in validation meta-analysis (S3). Data sets used in NSCLC histological subtype meta-analysis (S4). Gene-level results in NSCLC histological subtype (ADC vs SCC) meta-analysis (S5). Tissue microarray specimen annotations and PTK7 staining results (S6). Cell line annotations (S7).

XLS file - 68K, Onto-Express Analysis for Cellular Components

XLS file - 53K, Ingenuity Pathway Analysis for human-mouse orthologues

Lung cancer remains the most common cause of cancer-related death worldwide and it continues to lack effective treatment. The increasingly large and diverse public databases of lung cancer gene expression constitute a rich source of candidate oncogenic drivers and therapeutic targets. To define novel targets for lung adenocarcinoma, we conducted a large-scale meta-analysis of genes specifically overexpressed in adenocarcinoma. We identified an 11-gene signature that was overexpressed consistently in adenocarcinoma specimens relative to normal lung tissue. Six genes in this signature were specifically overexpressed in adenocarcinoma relative to other subtypes of non–small cell lung cancer (NSCLC). Among these genes was the little studied protein tyrosine kinase PTK7. Immunohistochemical analysis confirmed that PTK7 is highly expressed in primary adenocarcinoma patient samples. RNA interference–mediated attenuation of PTK7 decreased cell viability and increased apoptosis in a subset of adenocarcinoma cell lines. Further, loss of PTK7 activated the MKK7–JNK stress response pathway and impaired tumor growth in xenotransplantation assays. Our work defines PTK7 as a highly and specifically expressed gene in adenocarcinoma and a potential therapeutic target in this subset of NSCLC. Cancer Res; 74(10); 2892–902. ©2014 AACR.

Supplementary Methods and Materials from A Novel Lung Cancer Signature Mediates Metastatic Bone Colonization by a Dual Mechanism

PDF file - 19K, Decrease response to CAF-derived clcf1 in cntfr-depleted tumor cells

Abstract Locoregional failure (LRF) in patients with breast cancer post-surgery and post-irradiation is linked to a dismal prognosis. In a refined new model, we identified ectonucleotide pyrophosphatase/phosphodiesterase 1/CD203a (ENPP1) to be closely associated with LRF. ENPP1hi circulating tumor cells (CTC) contribute to relapse by a self-seeding mechanism. This process requires the infiltration of polymorphonuclear myeloid-derived suppressor cells and neutrophil extracellular trap (NET) formation. Genetic and pharmacologic ENPP1 inhibition or NET blockade extends relapse-free survival. Furthermore, in combination with fractionated irradiation, ENPP1 abrogation obliterates LRF. Mechanistically, ENPP1-generated adenosinergic metabolites enhance haptoglobin (HP) expression. This inflammatory mediator elicits myeloid invasiveness and promotes NET formation. Accordingly, a significant increase in ENPP1 and NET formation is detected in relapsed human breast cancer tumors. Moreover, high ENPP1 or HP levels are associated with poor prognosis. These findings unveil the ENPP1/HP axis as an unanticipated mechanism exploited by tumor cells linking inflammation to immune remodeling favoring local relapse. Significance: CTC exploit the ENPP1/HP axis to promote local recurrence post-surgery and post-irradiation by subduing myeloid suppressor cells in breast tumors. Blocking this axis impairs tumor engraftment, impedes immunosuppression, and obliterates NET formation, unveiling new opportunities for therapeutic intervention to eradicate local relapse and ameliorate patient survival. This article is highlighted in the In This Issue feature, p. 1171

The historical lack of preclinical models reflecting the genetic heterogeneity of multiple myeloma (MM) hampers the advance of therapeutic discoveries. To circumvent this limitation, we screened mice engineered to carry eight MM lesions (NF-κB, KRAS, MYC, TP53, BCL2, cyclin D1, MMSET/NSD2 and c-MAF) combinatorially activated in B lymphocytes following T cell-driven immunization. Fifteen genetically diverse models developed bone marrow (BM) tumors fulfilling MM pathogenesis. Integrative analyses of ∼500 mice and ∼1,000 patients revealed a common MAPK–MYC genetic pathway that accelerated time to progression from precursor states across genetically heterogeneous MM. MYC-dependent time to progression conditioned immune evasion mechanisms that remodeled the BM microenvironment differently. Rapid MYC-driven progressors exhibited a high number of activated/exhausted CD8+ T cells with reduced immunosuppressive regulatory T (Treg) cells, while late MYC acquisition in slow progressors was associated with lower CD8+ T cell infiltration and more abundant Treg cells. Single-cell transcriptomics and functional assays defined a high ratio of CD8+ T cells versus Treg cells as a predictor of response to immune checkpoint blockade (ICB). In clinical series, high CD8+ T/Treg cell ratios underlie early progression in untreated smoldering MM, and correlated with early relapse in newly diagnosed patients with MM under Len/Dex therapy. In ICB-refractory MM models, increasing CD8+ T cell cytotoxicity or depleting Treg cells reversed immunotherapy resistance and yielded prolonged MM control. Our experimental models enable the correlation of MM genetic and immunological traits with preclinical therapy responses, which may inform the next-generation immunotherapy trials.

Activating mutations in some isoforms of RAS or RAF are drivers of a substantial proportion of cancers. The main Raf effector, MEK1/2, can be targeted with several highly specific inhibitors. The clinical activity of these inhibitors seems to be mixed, showing efficacy against mutant BRAF-driven tumors but not KRAS-driven tumors, such as pancreatic adenocarcinomas. To improve our understanding of this context-dependent efficacy, we generated pancreatic cancer cells resistant to MEK1/2 inhibition, which were also resistant to KRAS and ERK1/2 inhibitors. Compared with parental cells, inhibitor-resistant cells showed several phenotypic changes including increased metastatic ability in vivo. The transcription factor SLUG, which is known to induce epithelial-to-mesenchymal transition, was identified as the key factor responsible for both resistance to MEK1/2 inhibition and increased metastasis. Slug, but not similar transcription factors, predicted poor prognosis of pancreatic cancer patients and induced the transition to a cellular phenotype in which cell-cycle progression becomes independent of the KRAS–RAF–MEK1/2–ERK1/2 pathway. SLUG was targeted using two independent strategies: (i) inhibition of the MEK5–ERK5 pathway, which is responsible for upregulation of SLUG upon MEK1/2 inhibition, and (ii) direct PROTAC-mediated degradation. Both strategies were efficacious in preclinical pancreatic cancer models, paving the path for the development of more effective therapies against pancreatic cancer. Significance: This study demonstrates that SLUG confers resistance to MEK1/2 inhibitors in pancreatic cancer by uncoupling tumor progression from KRAS–RAF–MEK1/2–ERK1/2 signaling, providing new therapeutic opportunities.

Few therapies are currently available for patients with KRAS-driven cancers, highlighting the need to identify new molecular targets that modulate central downstream effector pathways. Here we found that the microRNA (miRNA) cluster including miR181ab1 is a key modulator of KRAS-driven oncogenesis. Ablation of Mir181ab1 in genetically engineered mouse models of Kras-driven lung and pancreatic cancer was deleterious to tumor initiation and progression. Expression of both resident miRNAs in the Mir181ab1 cluster, miR181a1 and miR181b1, was necessary to rescue the Mir181ab1-loss phenotype, underscoring their nonredundant role. In human cancer cells, depletion of miR181ab1 impaired proliferation and 3D growth, whereas overexpression provided a proliferative advantage. Lastly, we unveiled miR181ab1-regulated genes responsible for this phenotype. These studies identified what we believe to be a previously unknown role for miR181ab1 as a potential therapeutic target in 2 highly aggressive and difficult to treat KRAS-mutated cancers.

Myeloid-derived suppressor cells (MDSCs) play a major role in cancer progression. In this study, we investigated the mechanisms by which complement C5a increases the capacity of polymorphonuclear MDSCs (PMN-MDSCs) to promote tumor growth and metastatic spread. Stimulation of PMN-MDSCs with C5a favored the invasion of cancer cells via a process dependent on the formation of neutrophil extracellular traps (NETs). NETosis was dependent on the production of high mobility group box 1 (HMGB1) by cancer cells. Moreover, C5a induced the surface expression of the HMGB1 receptors TLR4 and RAGE in PMN-MDSCs. In a mouse lung metastasis model, inhibition of C5a, C5a receptor-1 (C5aR1) or NETosis reduced the number of circulating-tumor cells (CTCs) and the metastatic burden. In support of the translational relevance of these findings, C5a was able to stimulate migration and NETosis in PMN-MDSCs obtained from lung cancer patients. Furthermore, myeloperoxidase (MPO)-DNA complexes, as markers of NETosis, were elevated in lung cancer patients and significantly correlated with C5a levels. In conclusion, C5a induces the formation of NETs from PMN-MDSCs in the presence of cancer cells, which may facilitate cancer cell dissemination and metastasis.

[Resumen] Antecedentes y objetivo. El alumnado con necesidades educativas especiales puede necesitar el apoyo específico y especializado de fisioterapia, con el fin de facilitar el desarrollo de su máximo potencial y ayudar en su inclusión educativa. La información acerca de la implementación de la fisioterapia en los centros educativos en el territorio español es muy escasa. El objetivo de este estudio es describir la situación de la fisioterapia en los centros educativos y compararla entre las diferentes comunidades autónomas. Materiales y métodos. Se realizó un estudio descriptivo y transversal a través de la elaboración de una encuesta online, diseñada junto a un panel de expertos a través del método Delphi. Se incluyeron seis bloques temáticos relacionados con la atención de fisioterapia, las condiciones laborales de los fisioterapeutas en los centros educativos y con la cobertura de las necesidades del alumnado. Resultados. Se obtuvieron 178 respuestas. Su distribución geográfica fue homogénea, aunque se detectaron grandes diferencias entre las comunidades autónomas. La mayoría de las personas encuestadas trabajan en centros de educación especial y existen comunidades autónomas en las que no se trabaja en centros ordinarios. Solo el 28,4% contestaron que la figura del fisioterapeuta está integrada en los equipos de orientación educativa de su comunidad autónoma y tienen la función de determinar la necesidad del recurso. Conclusiones. El desarrollo del trabajo de fisioterapia en los centros educativos como apoyo al alumnado con necesidades educativas especiales es desigual entre las comunidades autónomas, y en algunas, se considera insuficiente. [Abstract] Background and objective. Students with special educational needs may need the specific and specialized support of Physiotherapy in order to facilitate the development of their maximum potential and help in their educational inclusion. The information about school-based Physical Therapy development in Spain is very scarce. The objective of this study is to describe the situation of physiotherapy in educational centers and compare it among the different autonomous communities. Materials and methods. A descriptive and cross-sectional study was carried out through the elaboration of an online survey, designed together with a panel of experts using the Delphi method. Six thematic blocks were included related to the provision of the physiotherapy service, working conditions and the coverage of the students’ needs. Results. 178 responses were obtained. Their geographical distribution was homogeneous, although large differences were detected among the autonomous communities. Most of the participants work in special education centers and there are autonomous communities in which physical therapists do not work in ordinary centers. Only 28.4% answered that the physiotherapist is integrated into the educational orientation teams of their autonomous community and has the function of determining the need for the resource. Conclusions. School-based Physiotherapy services to support students with special educational needs is unequal among the autonomous communities, and in some of them it is considered insufficient.