FOXA1 mutations alter pioneering activity, differentiation and prostate cancer phenotypes.

Mutations in the transcription factor FOXA1 define a unique subset of prostate cancers but the functional consequences of these mutations and whether they confer gain or loss of function is unknown1-9. Here, by annotating the landscape of FOXA1 mutations from 3,086 human prostate cancers, we define two hotspots in the forkhead domain: Wing2 (around 50% of all mutations) and the highly conserved DNA-contact residue R219 (around 5% of all mutations). Wing2 mutations are detected in adenocarcinomas at all stages, whereas R219 mutations are enriched in metastatic tumours with neuroendocrine histology. Interrogation of the biological properties of wild-type FOXA1 and fourteen FOXA1 mutants reveals gain of function in mouse prostate organoid proliferation assays. Twelve of these mutants, as well as wild-type FOXA1, promoted an exaggerated pro-luminal differentiation program, whereas two different R219 mutants blocked luminal differentiation and activated a mesenchymal and neuroendocrine transcriptional program. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) of wild-type FOXA1 and representative Wing2 and R219 mutants revealed marked, mutant-specific changes in open chromatin at thousands of genomic loci and exposed sites of FOXA1 binding and associated increases in gene expression. Of note, ATAC-seq peaks in cells expressing R219 mutants lacked the canonical core FOXA1-binding motifs (GTAAAC/T) but were enriched for a related, non-canonical motif (GTAAAG/A), which was preferentially activated by R219-mutant FOXA1 in reporter assays. Thus, FOXA1 mutations alter its pioneering function and perturb normal luminal epithelial differentiation programs, providing further support for the role of lineage plasticity in cancer progression

A SMAD4-modulated gene profile predicts disease-free survival in stage II and III colorectal cancer

Objective: Loss of SMAD4 is associated with worse outcomes for colorectal cancer patients. We used gene ontology and bioinformatics to identify an RNA-based SMAD4-modulated profile and test its association with patient outcome. Design: Using a discovery dataset of 250 colorectal cancer patients, we analyzed expression of BMP/Wnt target genes for association with SMAD4 expression. Promoters of the BMP/Wnt genes were interrogated for SMAD-binding elements. 15 genes were implicated and three tested for modulation by SMAD4 in patient-derived colorectal cancer tumoroids. Expression of the 15 genes was used for unsupervised hierarchical clustering of a training dataset and two resulting clusters modeled in a centroid model. This model was applied to an independent validation dataset of stage II and III patients. Disease-free survival was analyzed by the Kaplan-Meier method. Results: In vitro analysis of three genes identified in the SMAD4-modulated profile (JAG1, TCF7, MYC) revealed modulation by SMAD4 consistent with the trend observed in the profile. In the training dataset (n = 553), the profile was not associated with outcome. However, among stage II and III patients (n = 461), distinct clusters were identified by unsupervised hierarchical clustering that were associated with disease-free survival (p = 0.02). The main model was applied to a validation dataset (n = 257) which confirmed the association of clustering with disease-free survival (p = 0.02). Conclusions: A SMAD4-modulated RNA-based gene profile identified high-risk stage II and III colorectal cancer patients, can predict disease-free survival, and has prognostic potential for stage II and III colorectal cancer patients

FFAR2 expressing myeloid-derived suppressor cells drive cancer immunoevasion

Abstract Background Emerging evidences suggest that aberrant metabolites contributes to the immunosuppressive microenvironment that leads to cancer immune evasion. Among tumor immunosuppressive cells, myeloid-derived suppressor cells (MDSCs) are pathologically activated and extremely immunosuppressive, which are closely associated with poor clinical outcomes of cancer patients. However, the correlation between MDSCs mediated immunosuppression and particular cancer metabolism remained elusive. Methods Spontaneous lung adenocarcinoma and subcutaneous mouse tumor models, gas chromatography–mass spectrometry (GC–MS) and immunofluorescence assay of patient-derived lung adenocarcinoma tissues, and flow cytometry, RNA sequencing and Western blotting of immune cells, were utilized. Results Metabolite profiling revealed a significant accumulation of acetic acids in tumor tissues from both patients and mouse model, which contribute to immune suppression and cancer progression significantly through free fatty acid receptor 2 (FFAR2). Furthermore, FFAR2 is highly expressed in the myeloid-derived suppressor cells (MDSCs) from the tumor of lung adenocarcinoma (LUAD) patients which is greatly associated with poor prognosis. Surprisingly, whole or myeloid Ffar2 gene deletion markedly inhibited urethane-induced lung carcinogenesis and syngeneic tumor growth with reduced MDSCs and increased CD8+ T cell infiltration. Mechanistically, FFAR2 deficiency in MDSCs significantly reduced the expression of Arg1 through Gαq/Calcium/PPAR-γ axis, which eliminated T cell dysfunction through relieving L-Arginine consumption in tumor microenvironment. Therefore, replenishment of L-Arginine or inhibition to PPAR-γ restored acetic acids/FFAR2 mediated suppression to T cells significantly. Finally, FFAR2 inhibition overcame resistance to immune checkpoint blockade through enhancing the recruitment and cytotoxicity of tumor-infiltrating T cells. Conclusion Altogether, our results demonstrate that the acetic acids/FFAR2 axis enhances MDSCs mediated immunosuppression through Gαq/calcium/PPAR-γ/Arg1 signaling pathway, thus contributing to cancer progression. Therefore, FFAR2 may serve as a potential new target to eliminate pathologically activated MDSCs and reverse immunosuppressive tumor microenvironment, which has great potential in improving clinical outcomes of cancer immunotherapy

A Tmprss2-CreERT2 Knock-In Mouse Model for Cancer Genetic Studies on Prostate and Colon.

Fusion between TMPRSS2 and ERG, placing ERG under the control of the TMPRSS2 promoter, is the most frequent genetic alteration in prostate cancer, present in 40-50% of cases. The fusion event is an early, if not initiating, event in prostate cancer, implicating the TMPRSS2-positive prostate epithelial cell as the cancer cell of origin in fusion-positive prostate cancer. To introduce genetic alterations into Tmprss2-positive cells in mice in a temporal-specific manner, we generated a Tmprss2-CreERT2 knock-in mouse. We found robust tamoxifen-dependent Cre activation in the prostate luminal cells but not basal epithelial cells, as well as epithelial cells of the bladder and gastrointestinal (GI) tract. The knock-in allele on the Tmprss2 locus does not noticeably impact prostate, bladder, or gastrointestinal function. Deletion of Pten in Tmprss2-positive cells of adult mice generated neoplasia only in the prostate, while deletion of Apc in these cells generated neoplasia only in the GI tract. These results suggest that this new Tmprss2-CreERT2 mouse model will be a useful resource for genetic studies on prostate and colon

Additional file 1 of FFAR2 expressing myeloid-derived suppressor cells drive cancer immunoevasion

Additional file 1. Supplementary figures S1–S9

Generation of the <i>Tmprss2-CreER</i>^<i>T2</i> knock-in mouse.

<p>(a) Schematic of targeting strategy. A cassette including an adenovirus splice acceptor (SA), followed by PGK-driven neomycin selection cassette flanked by FRT recombination sites, followed by the CreER^T2-IRES-nlsEGFP was used to replace exon 2 of mouse Tmprss2. The cassette is flanked by 3.5kb 5’ and 5kb 3’ homology arms. 5’ and 3’ Southern probes as was as HindIII (H) and EcoRI (E) sites and genotyping PCR primers (universal F1, wild-type specific R1, and knock-in specific R2) are depicted. The final transcript includes the non-coding exon 1 of Tmprss2 spliced into the CreER^T2-IRES-nlsEGFP gene. (b) Southern blot using 3’ probe and HindIII digestion. WT mice give a 7.5 kb band and the targeted mice (regardless of neomycin cassette) give a 8.2 kb band. (c) Southern blot using 5’ probe and EcoRI digestion. WT mice give a 9.5 kb band; the targeted mice with neomycin cassette (T) give a 9.9 kb band, while the mice with excised neomycin cassette (E) give a 9.5 kb band. (d) Genotype determination of wild-type and heterozygous mice by PCR. Wild-type fragment is 300-bp and mutant is 380-bp.</p

Prostate specific activity of <i>Tmprss2-CreER</i>^<i>T2</i> mice.

<p>(a) H&E and YFP IHC of anterior prostate, dorsolateral prostate, ventral prostate in TY mice. Scale bar represents 100 μM. (b) IF stain of basal cell marker P63 with endogenous YFP and DAPI fluorescence in TY mice. Scale bar represents 20 μM. (c) IF stain of luminal cell marker Ck8 with endogenous YFP and DAPI fluorescence in TY mice. Scale bar represents 20 μM. (d) Quantitative RT-PCR analysis of basal (<i>Ck5</i>, <i>Ck14</i>, <i>P63</i>) and luminal (<i>Ck8</i>, <i>Ck18</i>) marker expression in YFP+ and YFP- epithelial cells. Basal cell markers are strongly expressed YFP- cells; luminal cell markers strongly expressed in YFP+ cells. Expression was normalized to Actin. Results are shown as mean ± SD. (e) Comparison <i>of Tmprss2-CreER</i>^<i>T2</i> with <i>Nkx3</i>.<i>1-CreER</i>^<i>T2</i> driven conversion of membrane tdTomato (mT) to membrane EGFP (mG) of the anterior prostate. Scale bar represents 50 μM. (f) Same as in (e) but in periurethral proximal prostate. These cells are tightly packed with scant cytoplasm. The white line in the <i>Nkx3</i>.<i>1-CreER</i>^<i>T2</i> mouse separates the anterior prostate from periurethral prostate. Scale bar represents 50 μM.</p

Tissue distribution of <i>Tmprss2-CreER</i>^<i>T2</i> mediated recombination.

<p>(a) RNA-seq-based expression of <i>Tmprss2</i> mRNA in human tissues from the Genotype-Tissue Expression (GTEx) project. (b) MOE430-based expression of <i>Tmprss2</i> mRNA in mouse tissues from BioGPS. (c) Table of recombination efficiency, based on YFP IHC, in tissues of TY mice after tamoxifen administration. (d) H&E and YFP IHC of bladder and colon in TY mice. Scale bars represent 100 μM.</p

Tissue specific tumorigenesis of Apc deletion.

<p>(a) Characterization of Cre-mediated recombination in Tam-treated <i>Tmprss2CreER</i>^{<i>T2/ T2</i>}<i>; Apc</i>^{<i>LoxP/LoxP</i>} (TA) mice. Genomic DNA isolated from the indicated organs of a Tam-treated mouse were analyzed by PCR. The positions of the <i>Apc</i> floxed 498bp, and recombined 568bp DNA segments are indicated. (b) Gross pathology of colon of TA and wild-type mice following tamoxifen administration showing macroscopic colon polyps in tamoxifen treated mice. (c) H&E and β-catenin, Ki67 stain of TA and wild-type mouse colon. (d) H&E and β-catenin, Ki67 stain of TA and wild-type mouse prostate. Scale bars represent 100 μM.</p