The human Flower isoform hFWE4 facilitates cornification in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the second most common human tumor and arises from keratinocytes that comprise the epidermis and its associated hair follicles. Remarkably, transformed keratinocytes within a cSCC retain some ability to execute the terminal differentiation program that typically generates a stratified epidermis. Instead of the polarized stratification towards the body surface seen in normal epidermis, cSCC keratinocytes differentiate concentrically inward to produce keratin pearls. It is well appreciated that cSCC differentiation status is correlated with degree of local invasion and potential for distant metastasis, with well differentiated tumors having a better prognosis. While molecular mechanisms that govern the differentiation process are incompletely understood, identifying candidate molecules associated with cSCC differentiation can provide new histologic markers for prognostic stratification. Here we demonstrate that Flower (FWE), a newly described regulator of lamellar body (LB) trafficking in normal epidermis, is specifically expressed within highly differentiated layers of cSCC. Genetic knockout of the hFWE gene dysregulates cornification and LB-related gene expression leading to abnormal keratinization patterns, while ectopic hFWE4 expression drives G1-arrest and exit from the proliferative basal keratinocyte population. Additionally, we find that poorly differentiated keratinocyte regions of human cSCC exhibit minimal FWE-positivity, while this population is readily detectable in well-differentiated regions. We propose that FWE represents a novel differentiation marker in cSCC that could be utilized for classifying differentiation status and prognostic stratifying these tumors. Overall design: subcutaneous xenografts of hFWE WT or KO cells, 1*106 SCC13 cells were resuspended in 100µl of cold 1:1 sterile PBS:Matrigel (Corning 356237) and subsequently injected into the subcutaneous space on the dorsal flank of a NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mouse (Charles River 572). Tumors were harvested at 26d post injection for transcriptomic analysis.

皮肤鳞状细胞癌（cutaneous squamous cell carcinoma, cSCC）是人类第二高发的肿瘤，起源于构成表皮及其附属毛囊的角质形成细胞。值得注意的是，皮肤鳞状细胞癌中的转化角质形成细胞仍保留部分执行终末分化程序的能力，该程序通常可生成分层状表皮。与正常表皮中朝向体表的极性分层模式不同，皮肤鳞状细胞癌的角质形成细胞呈向内同心分化，进而形成角化珠。
学界已明确，皮肤鳞状细胞癌的分化状态与局部侵袭程度及远处转移潜能相关，分化良好的肿瘤预后更佳。尽管调控该分化过程的分子机制尚未完全阐明，但筛选与皮肤鳞状细胞癌分化相关的候选分子，可为肿瘤的预后分层提供新型组织学标志物。
本研究证实，Flower（FWE）——一种新近被报道的正常表皮板层小体（lamellar body, LB）运输调控因子——可特异性表达于皮肤鳞状细胞癌的高分化层中。对人源FWE（hFWE）基因进行基因敲除，会异常调控角质化及板层小体相关基因的表达，进而导致角化模式异常；而异位表达hFWE4则可促使细胞停滞于G1期，并退出增殖性基底角质形成细胞群体。
此外，本研究发现，人类皮肤鳞状细胞癌的低分化角质形成细胞区域几乎不表达FWE，而在高分化区域中则可轻易检测到该蛋白的阳性表达。我们据此提出，FWE可作为皮肤鳞状细胞癌的新型分化标志物，用于该肿瘤的分化状态分类与预后分层。
整体实验设计：将hFWE野生型（WT）或敲除型（KO）的SCC13细胞（1×10⁶个）重悬于100μl预冷的1:1无菌磷酸盐缓冲液（PBS）:基质胶（Matrigel，Corning 356237）混合液中，随后注射至NOD-Prkdcem26Cd52Il2rgem26Cd22/NjuCrl（NCG）小鼠（查尔斯河实验室，货号572）的背部皮下间隙。于注射后26天收获肿瘤，进行转录组学分析。