Publications

Abstract Breast cancer-associated fibroblasts (CAFs) have a crucial role in tumor initiation, metastasis and therapeutic resistance by secreting various growth factors, cytokines, protease and extracellular matrix components. Soluble factors secreted by CAFs are involved in many pathways including inflammation, metabolism, proliferation and epigenetic modulation, suggesting that CAF-dependent reprograming of cancer cells affects a large set of genes. This paracrine signaling has an important role in tumor progression, thus deciphering some of these processes could lead to relevant discoveries with subsequent clinical implications. Here, we investigated the mechanisms underlying the changes in gene expression patterns associated with the cross-talk between breast cancer cells and the stroma. From RNAseq data obtained from breast cancer cell lines grown in presence of CAF-secreted factors, we identified 372 upregulated genes, exhibiting an expression level positively correlated with the stromal content of breast cancer specimens. Furthermore, we observed that gene expression changes were not mediated through significant DNA methylation changes. Nevertheless, CAF-secreted factors but also stromal content of the tumors remarkably activated specific genes characterized by a DNA methylation pattern: hypermethylation at transcription start site and shore regions. Experimental approaches (inhibition of DNA methylation, knockdown of methyl-CpG-binding domain protein 2 and chromatin immunoprecipitation assays) indicated that this set of genes was epigenetically controlled. These data elucidate the importance of epigenetics marks in the cancer cell reprogramming induced by stromal cell and indicated that the interpreters of the DNA methylation signal have a major role in the response of the cancer cells to the microenvironment.

<p>Supplementary Figure 3</p>

No abstract is provided for this article.

<p>Study design participants, supplementary figure legends and List of cancer-related differentially methylated CpGs</p>

Abstract Background About 25% of Crohn’s disease (CD) patients develop perianal fistulizing disease (PFD), the pathogenesis of which remains poorly understood. Here we aimed to characterise the molecular and cellular events underlying this complication. Methods Non-inflamed rectal biopsies from CD patients with or without PFD (CD-PFD and CD) were processed for single-cell (n=14) or bulk (n=20) RNA analysis. Functional experiments were conducted on human primary intestinal fibroblasts and peripheral T cells. Results Single-cell RNA sequencing revealed the increased abundance of some cell types, such as intestinal fibroblasts, in the CD-PFD rectal mucosa relative to CD (Fig. 1A). Within the stromal subset, the lamina propria S1 fibroblasts showed the highest number of differentially expressed genes (DEGs) between CD and CD-PFD patients, including an upregulation of TFPI2 and a downregulation of the TGFβ-responding gene CTGF (Fig.1B). These changes were validated by bulk RNA analysis, where we also observed overexpression of the previously described PFD target genes MMP1 and MMP3 (Fig.1C). In vitro studies with intestinal fibroblasts confirmed the negative regulation by TGFβ of the PFD-signature, thus ruling out TGFβ as a driver of the PFD-associated fibroblast signature (Fig.1D). Next, we investigated DEGs in the lymphoid compartment and identified lymphotoxin beta (LTB) as being upregulated in CD-PFD intestinal CD4+ T cells (Fig.1E). LTB interacts with its receptor LTBR, which is expressed in stromal, epithelial and myeloid cells. Interestingly, LTB-LTBR interactions were increased in the CD-PFD group. In vitro studies in primary fibroblasts showed that LTB upregulates TFPI2, while it downregulates CTGF, thus reproducing the PFD-associated signature (Fig.1F). Finally, we submitted the top 50 genes overexpressed by CD-PFD CD4+ T cells to the immune dictionary1, which identified TL1A as its most likely driver (Fig.2A). In line with this, the TL1A receptor TNFRSF25 was overexpressed in CD-PFD CD4+ T cells (Fig.2B). As a link between TL1A and LTB is not described in the literature, we stimulated anti-CD3-activated peripheral T cells with TL1A and observed the significant upregulation of LTa1b2, the functional form of LTB (Fig.2C). Intriguingly, we also observed that while TNF can drive the PFD-associated signature in fibroblasts, it cannot induce LTa1b2 in T cells (Fig.2D), suggesting that TNF and TL1A/LTB signalling are two independent but potentially complementary pathways in PFD. Conclusion In summary, we propose a previously unrecognized TL1A-LTB axis as being responsible for inducing PFD-associated alterations in the rectal mucosa (Fig.2E), and we hypothesise that inhibition of TL1A might be worth exploring in the context of perianal disease. References 1. Cui A, Huang T, Li S, et al. Dictionary of immune responses to cytokines at single-cell resolution. Nature. 2024;625(7994):377-384.

Background Cancer of unknown primary ranks in the top ten cancer presentations and has an extremely poor prognosis. Identification of the primary tumour and development of a tailored site-specific therapy could improve the survival of these patients. We examined the feasability of using DNA methylation profiles to determine the occult original cancer in cases of cancer of unknown primary. Methods We established a classifier of cancer type based on the microarray DNA methylation signatures (EPICUP) in a training set of 2790 tumour samples of known origin representing 38 tumour types and including 85 metastases. To validate the classifier, we used an independent set of 7691 known tumour samples from the same tumour types that included 534 metastases. We applied the developed diagnostic test to predict the tumour type of 216 well-characterised cases of cancer of unknown primary. We validated the accuracy of the predictions from the EPICUP assay using autopsy examination, follow-up for subsequent clinical detection of the primary sites months after the initial presentation, light microscopy, and comprehensive immunohistochemistry profiling. Findings The tumour type classifier based on the DNA methylation profiles showed a 99·6% specificity (95% CI 99·5–99·7), 97·7% sensitivity (96·1–99·2), 88·6% positive predictive value (85·8–91·3), and 99·9% negative predictive value (99·9–100·0) in the validation set of 7691 tumours. DNA methylation profiling predicted a primary cancer of origin in 188 (87%) of 216 patients with cancer with unknown primary. Patients with EPICUP diagnoses who received a tumour type-specific therapy showed improved overall survival compared with that in patients who received empiric therapy (hazard ratio [HR] 3·24, p=0·0051 [95% CI 1·42–7·38]; log-rank p=0·0029). Interpretation We show that the development of a DNA methylation based assay can significantly improve diagnoses of cancer of unknown primary and guide more precise therapies associated with better outcomes. Epigenetic profiling could be a useful approach to unmask the original primary tumour site of cancer of unknown primary cases and a step towards the improvement of the clinical management of these patients. Funding European Research Council (ERC), Cellex Foundation, the Institute of Health Carlos III (ISCIII), Cancer Australia, Victorian Cancer Agency, Samuel Waxman Cancer Research Foundation, the Health and Science Departments of the Generalitat de Catalunya, and Ferrer.

Abstract Disruption of circadian rhythms, daily oscillations in biological processes that are regulated by an endogenous clock, has been linked to tumorigenesis. Normal and malignant tissues often show asynchronies in cell proliferation and metabolic rhythms. Cancer chronotherapy takes biological time into account to improve the therapy. However, alterations of the circadian clock machinery genes have rarely been reported in human cancer. Herein, we show that the BMAL1 gene, a core component of the circadian clock, is transcriptionally silenced by promoter CpG island hypermethylation in hematologic malignancies, such as diffuse large B-cell lymphoma and acute lymphocytic and myeloid leukemias. We also describe how BMAL1 reintroduction in hypermethylated leukemia/lymphoma cells causes growth inhibition in colony assays and nude mice, whereas BMAL1 depletion by RNA interference in unmethylated cells enhances tumor growth. We also show that BMAL1 epigenetic inactivation impairs the characteristic circadian clock expression pattern of genes such as C-MYC, catalase, and p300 in association with a loss of BMAL1 occupancy in their respective promoters. Furthermore, the DNA hypermethylation–associated loss of BMAL1 also prevents the recruitment of its natural partner, the CLOCK protein, to their common targets, further enhancing the perturbed circadian rhythm of the malignant cells. These findings suggest that BMAL1 epigenetic inactivation contributes to the development of hematologic malignancies by disrupting the cellular circadian clock. [Cancer Res 2009;69(21):8447–54]

Background: Neuroblastoma is the most common extracranial solid malignancy in infancy and childhood, but the biological factors involved in its development and progression are still unclear. Transcriptional silencing of tumor suppressor genes mediated by hypermethylation of promoter CpG islands is a hallmark of human tumors. We addressed the clinical relevance of promoter hypermethylation in neuroblastoma. Methods: We examined the methylation status of 45 candidate genes representative of many cellular pathways in 10 neuroblastoma cell lines and of 10 of these genes in 145 tumor samples (118 of them were primary neuroblastomas). We used Fisher's exact test to examine the association of CpG island methylation and clinical subgroups and Kaplan–Meier analysis to determine the association between methylation and survival in primary tumors. Cluster analysis was used to group cell lines and tumors by gene methylation status. Bonferroni-corrected statistical tests were two-sided. Results: Clustering of neuroblastoma cell lines on the basis of hypermethylation distinguished lines with MYCN amplification (a negative prognostic factor) from those without it ( P = .012). Promoter hypermethylation of the developmental gene HOXA9 was associated with mortality in noninfant patients ( P = .04) and in tumors lacking MYCN amplification ( P = .023). Hypermethylation of the proapoptotic gene TMS1 and the cell cycle gene CCND2 was associated with stage 4–progressing tumors ( P <.001), but the genes were never methylated in stage 4S tumors, which undergo spontaneous regression. Hypermethylation of the differentiation gene RARβ2 was associated with patient survival ( P = .032). Unsupervised hierarchical cluster analysis of all tumors based on methylation of the 10 genes separated several clinically relevant groups of tumors. Conclusions: Profiling the status of CpG island hypermethylation in human primary neuroblastomas may have clinicopathologic value.

&lt;p&gt;Supplementary Tables S1-S7. K-RasG12V tumors serially transplanted in Crl:NU-Foxn1nu mice and paired derived cell lines (S1); Semiquantitative assessment of the percentages of histologic components in passage #1 engrafted tumors (S2); EML4-ALK tumors orthotopically implanted in Crl:NU-Foxn1nu mice (S3); Histologic progression of orthoallografts upon sequential mice-to-mice passages (S4); Presence of distal metastasis in animals implanted with the indicated orthoallografts (S5); Summary of primary cell lines obtained from the indicated orthoallograft implants (S6); Histopathological characterization of EML4-ALK implants after crizotinib treatment (S7).&lt;/p&gt;

&lt;p&gt;Mutational status of the lymphoid compartment&lt;/p&gt;

&lt;p&gt;Distribution of clinical and pathological features in the validation cohort BR1301a&lt;/p&gt;

Summary Methylation in the promoter region of many genes is involved in regulating gene expression patterns. Using the Illumina GoldenGate © methylation assay, we examined the methylation status of 1505 CpG‐sites from 807 genes in 32 samples from patients with acute myeloid leukaemia (AML) at diagnosis, nine at relapse and 15 normal controls and performed additional pyrosequencing and semiquantitative methylation specific polymerase chain reaction (MSP) of the GNMT promoter in 113 diagnostic AML samples. We found a gain of overall methylation in AML samples with a further increase at relapse. Regional hypermethylation as assessed by array analysis could be confirmed by both MSP and pyrosequencing. Additionally, large‐scale methylation analysis identified interesting candidate genes. Cluster analysis indicated that cytogenetic subgroups seemed to be characterized by additional distinct epigenetic modifications and that basic DNA methylation patterns remain at relapse. Therefore, promoter hypermethylation is a frequent event in AML and is accentuated at relapse. Array‐based methylation analysis determined distinct methylation profiles for non‐malignant controls and AML samples with specific chromosomal aberrations and can identify target genes for further evaluation.