SPARC aberrant methylation in Idiopathic pulmonary fibrosis: an explorative study

Federico Pio Fabrizio^1*Angelo Sparaneo²Flavia Centra²Francesco Delli Muti²Paola Parente³MASSIMILIANO COPETTI⁴Marco Donatello Delcuratolo⁵Antonio Rossi⁶Elisa Gili⁷Giulio Rossi⁸Paolo Graziano³Lucia Anna Muscarella^2*

• ¹Department of Medicine and Surgery, Kore University of Enna, Enna, Italy
• ²Laboratory of Oncology, Fondazione Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo, Italy
• ³Unit of Pathology, Fondazione Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo, Italy
• ⁴Unit of Biostatistic, Fondazione Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo, Italy
• ⁵Unit of Oncology, Fondazione Casa Sollievo della Sofferenza IRCCS, San Giovanni Rotondo, Italy
• ⁶Oncology Centre of Excellence, Therapeutic Science & Strategy Unit, IQVIA,, Milan, Italy
• ⁷Department of Clinical and Experimental Medicine, Universita degli Studi di Catania, Catania, Italy
• ⁸Pathologic Anatomy Unit, Azienda Ospedaliero-Universitaria di Modena, Modena, Italy

Idiopathic pulmonary fibrosis (IPF) is a chronic interstitial lung disease (ILD) characterized by progressive accumulation of extracellular matrix in the lung and dysregulated activation of specific signaling pathways. Recent advances in the understanding of the biological bases of IPF identified the silencing of Secreted protein acidic and rich in cysteine (SPARC) as a key modulator in the pathogenesis of IPF, although the mechanisms underlying the SPARC aberrant modulation remain to be fully elucidated. Here we investigated the aberrant methylation at the promoter gene region as a possible mechanism of SPARC deregulation in IPF. Formalin-fixed paraffin-embedded (FFPE) tissues from a cohort of 44 patients with IPF and from a control-group of 23 non-idiopathic pulmonary fibrosis (NIPF) were analyzed. DNA methylation analysis at the SPARC promoter region was assessed by quantitative methylation-specific PCR analysis (QMSP) and a total of 11 CpGs located in the gene promoter island were evaluated. Methylation levels were found to be significantly higher (p<0.004, Mann-Whitney test) in 44 IPF samples (methylated using the optimal cut-off 20/44, 45%) compared to NIPF surgical biopsies (methylated using the optimal cut-off 3/23, 13%). At the in vitro level, we observed an inverse correlation between SPARC mRNA levels and hypermethylation under 5-Aza-2′deoxycytidine (5-Aza-CdR) treatment when a primary fibrotic cell line was treated, whereas any variations were observed treating non-fibrotic cells. Our explorative study suggests that promoter methylation of the SPARC gene is linked to IPF but not to NIPF, and could represent a potential molecular marker of disease, thus warranting further investigations on larger cohorts.