Degree of Tissue Differentiation Dictates Susceptibility to BRAF-driven Colorectal Cancer. Degree of Tissue Differentiation Dictates Susceptibility to BRAF-driven Colorectal Cancer

In murine models, we find that oncogenic BRAF paradoxically suppresses stem cell renewal and instead promotes differentiation and senescence. This corresponds to inefficient tumor formation in oncogenic BRAF mouse models of colon cancer. By reducing levels of differentiation in the gut via genetic manipulation of either of two distinct differentiation-promoting factors (Smad4 or Cdx2), stem cell activity is restored in BRAFV600E intestines, and the oncogenic capacity of mutant BRAF is amplified. In human patients, we observe that reduced levels of differentiation in normal tissue is associated with increased susceptibility to serrated colon tumors. Together, these findings help resolve the conditions necessary for BRAF-driven colon cancer initiation. Overall design: RNAs were extracted from crypt jejunal epithelia of the indicated genotypes (BRAF-V600Ef/+;Villin-CreER(T2), Smad4f/f BRAF-V600Ef/+;Villin-CreER(T2) and Cdx2f/f BRAF-V600Ef/+Villin-CreER(T2)) one day following 4 consecutive days of tamoxifen injection. Uninjected mice served as control. After flushing the freshly harvested jejunum with cold PBS, the epithelia was dissociated from underlying mesenchyme by incubating with 3 mM EDTA/PBS at 4°C as described previously (Perekatt, 2014), and filtered through 70 micron filter to isolate the crypts. The crypts were washed with PBS, and pelleted to remove excess PBS prior to addition of TRIzol for RNA extraction according to manufacturer’s protocols.

在小鼠模型中，我们发现致癌性BRAF（oncogenic BRAF）会反常地抑制干细胞自我更新，转而促进细胞分化与衰老。这与致癌性BRAF结肠癌小鼠模型中肿瘤生成效率低下的现象相一致。通过对两种不同的促分化因子（Smad4或Cdx2）进行基因操作以降低肠道中的分化水平，BRAFV600E突变小鼠肠道内的干细胞活性得以恢复，突变BRAF的致癌能力也得到增强。在人类患者中，我们观察到正常组织的分化水平降低与锯齿状结肠肿瘤的易感性升高相关。综上，这些研究结果阐明了BRAF驱动的结肠癌起始所必需的条件。

实验整体设计：
在连续4天注射他莫昔芬（tamoxifen）后的第1天，从指定基因型的空肠隐窝上皮细胞中提取RNA，所用基因型包括：BRAF-V600Ef/+;Villin-CreER(T2)、Smad4f/f BRAF-V600Ef/+;Villin-CreER(T2)以及Cdx2f/f BRAF-V600Ef/+Villin-CreER(T2)。未注射他莫昔芬的小鼠作为对照。将新鲜采集的空肠用预冷的磷酸盐缓冲液（Phosphate Buffered Saline，PBS）冲洗后，按照此前的方法（Perekatt等，2014），通过在4℃下与3 mM乙二胺四乙酸（EDTA）/PBS孵育，将上皮细胞与下方的间充质分离，随后经70微米滤膜过滤以分离隐窝。将分离得到的隐窝用PBS洗涤并离心沉淀以去除多余PBS，随后按照试剂盒说明书加入TRIzol进行RNA提取。