PO.MCB11.02 · 分子与细胞生物学
人类Flower异构体hFWE4促进皮肤鳞状细胞癌中的角化
The human Flower isoform hFWE4 facilitates cornification in cutaneous squamous cell carcinoma
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
背景:皮肤鳞状细胞癌(cSCC)起源于转化的角质形成细胞,这些细胞常保留部分分化潜能。由于分化状态与预后相关,区分高分化与低分化(WD vs PD)肿瘤的分子标志物具有临床价值。Flower(FWE)是一种四次跨膜蛋白,在表皮屏障形成过程中调节板层小体(LB)运输,本研究将其作为cSCC终末分化的候选标志物和调节因子进行考察。
方法:分析了FWE在人类cSCC细胞系、异种移植瘤和临床肿瘤中的表达。使用CRISPR/Cas9生成hFWE敲除(KO)SCC-13细胞,用于在NCG小鼠中进行异种移植(n=12)。通过RNA-seq、免疫印迹和免疫荧光对KO肿瘤中与分化相关的变化进行定量。使用慢病毒EGFP-2A-hFWE4构建体评估hFWE4过表达对增殖和分化结局的影响。
结果:FWE在培养的SCC细胞Ca²⁺驱动的分化过程中被诱导,并定位于SCC-13异种移植瘤中的基底上层角质形成细胞。hFWE KO肿瘤的平均质量略有下降(从0.33降至0.19g,p<0.05),并表现出角化改变,其特征为含透明角质颗粒的细胞减少和实性角化不全。KO肿瘤的免疫荧光显示filaggrin阳性和loricrin阳性区域分别减少了63%和82%(p<0.01-0.0001),而RNA-seq鉴定出73个下调基因(FDR<0.05,|log₂FC|>1)——富集于LB和角化通路——包括KLK5、KLK7、SLURP1和LORICRIN。异位hFWE4表达在SCC-13、COLO 16和SCC-12b.2细胞中诱导了G1期阻滞(G1期增加5-17%,S期减少6-10%;p<0.0001),并在SCC-13异种移植瘤中降低了Ki67⁺细胞的比例(从0.57%降至0.13%,p<0.05)和ITGB1⁺细胞的比例(从11.3%降至2.4%,p<0.05),同时增加了filaggrin⁺细胞的比例(从34.6%增至57.2%;p<0.01)。在人类肿瘤中(WD n=9,PD n=5),WD区域的FWE阳性面积显著高于PD区域(增加6.2倍;p<0.0001)。
结论:在cSCC中,FWE缺失破坏了LB依赖的角化,而异位表达则引发G1期阻滞和分化。由于高FWE阳性面积与人类cSCC分化等级升高相关,我们提出FWE既是角化的机制性调节因子,也是客观分级cSCC分化的有前景的分子标志物。
查看英文原文 English abstract
Background: Cutaneous squamous cell carcinoma (cSCC) arises from transformed keratinocytes that often retain partial differentiation potential. Because differentiation status correlates with prognosis, molecular markers that distinguish well- from poorly-differentiated (WD vs PD) tumors are of clinical value. Flower (FWE), a four-transmembrane protein that regulates lamellar body (LB) trafficking during epidermal barrier formation, was examined here as a candidate marker and regulator of terminal differentiation in cSCC.
Methods: FWE expression was analyzed in human cSCC cell lines, xenografts, and clinical tumors. CRISPR/Cas9 was used to generate hFWE knockout (KO) SCC-13 cells for xenografting in NCG mice (n=12). RNA-seq, immunoblotting, and immunofluorescence quantified differentiation-associated changes in KO tumors. Lentiviral EGFP-2A-hFWE4 constructs were used to assess impact of hFWE4 overexpression on proliferation and differentiation outcomes.
Results: FWE was induced during Ca²⁺-driven differentiation of cultured SCC cells and localized to suprabasal keratinocytes in SCC-13 xenografts. hFWE KO tumors exhibited a slight reduction in average mass (0.33 to 0.19g, p<0.05) and showed altered keratinization characterized by reduced keratohyalin granule-containing cells and solid parakeratosis. Immunofluorescence of KO tumors revealed 63% and 82% reductions in filaggrin- and loricrin-positive areas, respectively (p<0.01-0.0001), while RNA-seq identified 73 downregulated genes (FDR<0.05, |log₂FC|>1)-enriched for LB and cornification pathways-including KLK5 , KLK7 , SLURP1 , and LORICRIN . Ectopic hFWE4 expression induced G1 arrest (↑G1 by 5-17%, ↓S-phase by 6-10%; p < 0.0001) in SCC-13, COLO 16 and SCC-12b.2 cells, and reduced the fraction of cells that were Ki67⁺ (0.57% to 0.13%, p<0.05) and ITGB1⁺ (11.3% to 2.4%, p<0.05) while increasing the fraction that were filaggrin<⁺ (34.6% to 57.2%; p<0.01) in SCC-13 xenografts. In human tumors (WD n=9, PD n=5), FWE-positive area was significantly higher in WD than PD regions (6.2-fold increase; p<0.0001).
Conclusions: In cSCC, loss of FWE disrupts LB-dependent cornification, while ectopic expression elicits G1-arrest and differentiation. As high FWE-positive area correlates with increased differentiation grade in human cSCC, we propose that FWE represents both a mechanistic regulator of cornification and a promising molecular marker for objective grading of cSCC differentiation.
利益披露 Disclosure
J. C. Rudd, None..
P. T. Kuwong, None..
R. E. Johnson, None..
L. N. Monga-Wells, None..
M. Vo, None..
J. Russolillo, None..
S. Reddy, None..
M. Jacob, None..
H. Litz, None..
C. Li, None..
A. Siref, None..
J. A. Grunkemeyer, None..
L. A. Hansen, None.