Schlafen 12 Interaction with SerpinB12 and Deubiquitylases Drives Human Enterocyte Differentiation

Abstract

Background/Aims: Human enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation. Methods: We used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells. Results: Fasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes. Conclusions: SLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.

Schlafen 12 Is Prognostically Favorable and Reduces C-Myc and Proliferation in Lung Adenocarcinoma but Not in Lung Squamous Cell Carcinoma

“…Our mRNA level analysis excluded transcriptional inhibition of c-myc by SLFN12 ( Figure 6 A), as SLFN12 did not decrease c-myc mRNA, and indeed seemed to increase it in two of the three cell lines. Next, because SLFN12 regulates ZEB1 in breast cancer through translational inhibition [ 12 ], and CDX2 in intestinal epithelial cells by modulating the deubiquitylases USP14 and UCHL5 [ 11 ], we decided to separately inhibit the proteasome and UCHL5/USP14 deubiquitylase activity to determine if either would block the ability of SLFN12 to reduce c-myc protein. However, neither proteasomal inhibition nor inhibition of these deubiquitylases blocked SLFN12 regulation of c-myc ( Figure 6 B–E).…”

“…This study identified SLFN12 as prognostically favorable in LUAD, but not in LUSC. Although SLFN12 has previously been shown to regulate differentiation in other epithelial cell types [ 11 , 12 , 13 ], SLFN12 changed mRNA levels of differentiation markers (SCGB1A1, SFTPC, HOPX, CDH1, CK-5, and P63) in a complex and inconsistent fashion among LUAD and LUSC cell lines. However, SLFN12 tended to correlate differently with myc-associated gene signatures between human lung adenocarcinomas and human lung squamous cell carcinomas, while SLFN12 reduced both c-myc protein levels and cell proliferation only in lung adenocarcinoma cell lines, with no similar effects in LUSC.…”

“…Furthermore, SLFN11 and SLFN5 are long Schlafens that possess a nuclear import signal and a putative RNA helicase domain, and bind directly to chromosomal DNA. In contrast, the intermediate SLFN12 lacks such sequences and is localized to the cytoplasm [ 11 , 26 ]. Therefore, one would not necessarily expect SLFN12 to act in the same fashion as the long Schlafen proteins.…”

“…SLFN12 would thus not be expected to act in the same fashion as a long Schlafen. Our previously published work identified SLFN12 as modulating differentiation in intestinal epithelium, breast cancer, and prostate cancer [ 11 , 12 , 13 ]. We now want to understand whether SLFN12 plays a major role in lung cancer biology and prognosis and whether this role might differ between adenocarcinoma and squamous carcinoma of the lung.…”

Loss of Schlafen3 influences the expression levels of Schlafen family members in ileum, thymus, and spleen tissue

“…Indeed, this occurs even when the entry of the over expressed Slfn3 into the nucleus is prevented by adding a nuclear exclusion sequence (Chaturvedi et al, 2014). Cytosolic SLFN12, which is the likely human ortholog of Slfn3, regulates human enterocytic differentiation via a pathway involving SerpinB12 and the deubiquitylases USP14 and UCHL5 to change expression of the transcription factor Cdx2 which in turn modulates human gene expression (Basson et al, 2018). Such cytosolic modulation of gene expression Slfn3 could modulate the expression of other Slfn family members through such pathways.…”