FAM13A as potential therapeutic target in modulating TGF-beta-induced airway tissue remodeling in COPD.

Genome-wide association studies have shown that a gene variant in the Family with Sequence Similarity 13, Member A (FAM13A) is strongly associated with reduced lung function and the appearance of respiratory symptoms in patients with Chronic Obstructive Pulmonary Disease (COPD). A key player in smoking-induced tissue injury and airway remodeling is the transforming growth factor β1 (TGFβ1). To determine the role of FAM13A in TGFβ1 signaling, FAM13A-/- airway epithelial cells were generated using CRISPR-Cas9, while over-expression of FAM13A was achieved using lipid nanoparticles. Wildtype (WT) and FAM13A-/- cells were treated with TGFβ1, followed by gene and/or protein expression analyses. FAM13A-/- cells augmented TGFβ1-induced increase in COL1A1 and MMP2 expression compared to WT cells. This effect was mediated by an increase in CTNNB1 expression in FAM13A-/- cells compared to WT cells after TGFβ1 treatment. FAM13A over-expression was partially protective from TGFβ1-induced COL1A1 expression. Finally, we showed that airway epithelial-specific FAM13A protein expression is significantly increased in patients with severe COPD compared to control non-smokers, and negatively correlated with lung function. In contrast, β-catenin (CTNNB1), which has previously been linked to be regulated by FAM13A, is decreased in the airway epithelium of smokers with COPD compared to non-COPD subjects. Together, our data showed that FAM13A may be protective from TGFβ1-induced fibrotic response in the airway epithelium via sequestering CTNNB1 from its regulation on downstream targets. Therapeutic increase in FAM13A expression in the airway epithelium of smokers at risk for COPD, and those with mild COPD, may reduce the extent of airway tissue remodeling.


Introduction
In this study, we used CRISPR-Cas9 gene editing technology in airway epithelial cells to 86 determine the effects of FAM13A on TGFβ1-mediated response, and characterize FAM13A 87 protein expression in human lung tissues from individuals with and without COPD. First, we 88 showed that loss of FAM13A increased TGFβ1-induced rise in COL1A1, MMP2 and CTNNB1

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Human lung tissues for immunohistochemistry 101 Formalin-fixed paraffin-embedded (FFPE) human lung tissue samples were obtained from five 102 non-smoking controls, nine smokers with COPD, with Global initiative for chronic obstructive 103 Lung Disease (GOLD) stage 2 (moderate) disease and eight smokers with GOLD3 or 4 (severe or 104 very severe). All of these samples were collected following informed consent from patients 105 undergoing thoracic surgery who donated lung tissues to the UBC James Hogg lung tissue registry

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All of these studies were pooled into one cohort as these studies were generated using the same 124 microarray platform (U133A Plus 2.0 array) and reported by the same lab. Raw data (.CEL files) 125 downloaded from the GEO were tested for batch effect and outliers using principal component 126 analysis. Since no obvious batch effect was present, the combined data were normalized using the 127 Robust Multi-array Average (RMA) method. After confirmation and removal of three outliers, this 128 cohort contains 68 non-smokers, 101 smokers and 42 smokers with COPD. A summary of the 129 patient demographics is presented in Table 2. previously reported (48) and presented below in Table 3.

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Immunofluorescence staining and confocal microscopy 196 To visualize COL1A1 and CTNNB1 protein expression, cells were fixed with formalin,  Fluorescence intensity from the entire image was normalized to nuclei count using Image J.   Coulter) was used to analyze the % GFP+ and DiD+ cells in the samples.

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Cell imaging 239 To observe single cell dynamics in real time, cells were seeded at low density in 10% FBS DMEM 240 and allowed to attach for 5h. Fresh complete media was replaced prior to live cell imaging every 241 min for 3h. To observe changes in cellular morphology relative to density over time, WT and 242 FAM13A -/-1HAEo cells were seeded at low density in 10% FBS DMEM with fresh complete 243 media containing TGFβ1 replaced every 2 days.

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In-vitro and ex-vivo study data were tested for normality prior to the selection of a parametric

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Expression of FAM13A is increased and CTNNB1 is decreased in the airway epithelium of 256 patients with COPD. 257 To determine the relationship between FAM13A and CTNNB1 expression in airway epithelium, 258 FAM13A expression in lung tissue samples from non-smoking controls and subjects with COPD 259 was evaluated by immunohistochemistry. The demographics of this cohort is shown in Table 1.

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Representative images of airway epithelial-specific FAM13A protein expression in non-smoking 261 controls and COPD patients with GOLD 2 and GOLD 4 are shown in Figure 1A-C. FAM13A 262 expression was significantly increased in the airway epithelia of patients with moderate to severe 263 COPD (as defined by GOLD Stages 3 & 4) when compared to those from non-smoking controls 264 ( Figure 1D). After stratification of COPD patients by smoking status, epithelial-specific FAM13A 265 expression was highest in current smokers when compared to non-smoking controls and 266 moderately elevated in ex-smokers ( Figure 1E). Interestingly, we observed a significant negative

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In a replication cohort (Table 3)

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To determine the biological role of FAM13A in response to TGFβ1 stimulation, we used CRISPR-

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Active CTNNB1 expression is elevated in FAM13A -/cells and further induced by TGFβ1. 308 Previously, it was reported that a loss of FAM13A increases the stability and expression of 309 CTNNB1, which protects mice from chronic smoke-induced emphysema (27). Since CTNNB1 is 310 also a transcriptional co-regulator that is involved in EMT (6)

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Given that reduced FAM13A expression led to upregulation of COL1A1 and MMP2 expression 344 via TGFβ1, we determined whether the over-expression of FAM13A reverses this phenomenon.

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Initially, we used lipofectamine to transfect cells with FAM13A mRNA, and showed that FAM13A 346 over-expression attenuated TGFβ1-induced increase in COL1A1 but not MMP2 gene expression 347 (Supplement Figure 3A-C) (https://figshare.com/s/9d1590d6aba3fbe399e4). Due to its inherent 348 toxicity and its ability for immune activation, lipofectamine has little clinical utility. Therefore, we 349 next evaluated the therapeutic potential of FAM13A over-expression using clinically-validated 350 LNP technology (34, 58). We encapsulated mRNA in LNP for cellular over-expression. Using human subjects, we found that FAM13A protein expression was upregulated, while CTNNB1 gene 407 expression was downregulated, in smokers with COPD compared to healthy non-smokers.

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Collectively, these data suggest that FAM13A is a modifier of TGFβ1-mediated signaling and its 409 up-regulation in smokers at risk for COPD may protect the lungs against epithelial remodeling that 410 leads to progressive narrowing of the airways in COPD.

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Mass spectrometry studies of FAM13A-interacting proteins have implicated pathways enriched in 413 "CTNNB1 phosphorylation cascade" and "CTNNB1 degradation by the destruction complex"  The exact mechanism by which FAM13A is upregulated in the airway epithelium of patients with 488 COPD is unknown and awaits resolution. However, we speculate that upregulation of FAM13A 489 in the airway epithelium of healthy smokers without COPD may reduce CTNNB1 expression for 490 a more protective response in controlling excess tissue remodeling. Undoubtedly, CTNNB1 (β-491 catenin) signaling contributes to lung repair/regeneration in response to various injuries (24, 31); 492 therefore, more extensive studies accounting for multiple competing mechanisms at play are 493 required to determine the value of over-expressing FAM13A in tissue-and disease-specific 494 treatment.

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In summary, this report shows that airway epithelial cells deficient in FAM13A loses its ability to Acknowledgments 508 509 We thank Amrit Samra for processing the lung tissue samples for immunohistochemical staining, 510 and Dean English for scanning the sections using the Aperio Imaging System.