并非可靠的一个"抑癌基因"：脂质磷酸酶INPP4B促进结直肠癌细胞增殖及对EGFR单抗治疗耐受的研究

Although one of the 4-phosphatases, 4-phosphatase type II (INPP4B), has been shown to inhibit PI3K signalling and have a tumor suppressive role in some other types of cancers, we found in preliminary studies that, strikingly, INPP4B was frequently upregulated in colon cancer. In addition, our results showed that INPP4B contributed to constitutive activation of Akt and played a role in resistance to the anti-EGFR antibody panitumumab in colon cancer cells, in particular, in those carrying wild-type KRAS, and was associated with poor prognosis of colon cancer patients. Analysis of published gene array datasets revealed that the INPP4B transcript was similarly elevated in a subset of colon cancer, which was also associated with poor patient prognosis. In this project, we will test the hypotheses that: 1), INPP4B upregulation confers a survival and proliferation advantage to colon cancer cells; and 2), elevated INPP4B is a predictive biomarker of responses of wild-type KRAS colon cancer to treatment with anti-EGFR antibodies. The specific aims are: to determine whether INPP4B enhances colon cancer cell proliferation and survival; to define the mechanisms by which INPP4B promotes Akt activation and resistance to anti-EGFR antibodies; to determine the effect of INPP4B on colon cancer growth and responses to anti-EGFR antibodies in vivo; to determine whether elevated INPP4B is predictive of poor outcome of colon cancer patients treated with anti-EGFR antibodies; and to identify the mechanism of INPP4B upregulation in colon cancer cells. This project will identify a hitherto unrecognized mechanism that promotes PI3K signaling in colon cancer cells, and define INPP4B as a proliferation and prosurvival protein in colon cancer, in contrast to its function as a tumor suppressor in some other types of cancers. The results will provide proof-of-principle for development of inhibitors targeting INPP4B in the treatment of colon cancer. Identification of elevated INPP4B as a predictive biomarker of poor outcome of patients with EGFR-expressing wild-type KRAS colon cancer treated with anti-EGFR antibodies will also be instructive for refining personalized targeted therapy in the treatment of colon cancer.

作为一个 PI3K信号的负调节因子， 脂质磷酸酶INPP4B在一些组织中起抑癌作用。但我们先期研究发现INPP4B 在结肠癌中常呈高表达，并和患者的不良预后有关；shRNA敲低INPP4B的表达抑制结肠癌细胞中PI3K下游激酶Akt和SGK3的活化，并增强其对EGFR 单抗的敏感性。因此我们推论：1）INPP4B在结肠癌中增强PI3K信号的活化；2）INPP4B促进结肠癌细胞的增殖及存活；3）INPP4B 的高表达可作为一种预测野生型KRAS 结肠癌对EGFR单抗治疗耐受的分子标志。本研究将利用诱导shRNA敲低INPP4B表达的结肠癌细胞株，以同样处理的乳腺癌细胞株为对照，确认其在结肠癌细胞中特有的增强PI3K信号的活化及促进细胞增殖和存活的功能，并在人结肠癌小鼠移植瘤模型予以验证。同时，采用免疫组化检测结肠癌组织INPP4B的表达，结合临床数据确定其高表达和EGFR单抗治疗效果的关系。

磷脂酰肌醇-4-磷酸酶（Inositol polyphosphate 4-phosphatase type II， INPP4B）在多种肿瘤中负向调节PI3K信号通路，发挥着抑癌基因的作用。但是，我们发现其在结肠癌中上调。通过敲低INPP4B，阻止了AKT和SGK3的激活，抑制了结肠癌细胞的增殖，阻滞了结肠癌异种移植瘤生长。反过来，过表达INPP4B，增加了正常结肠上皮的增殖，触发了正常结肠上皮的独立锚定生长。而且，我们证明了INPP4B通过其蛋白磷酸酶活性去磷酸化PTEN，使得PTEN在Ser380,Thr382,Thr383,Ser385这一簇位点的磷酸化水平降低，导致PTEN稳定性降低，生物活性功能下降，从而提高细胞内PI(3,4,5)P3水平，激活PI3K/Akt/SGK3信号通路。对INPP4B在结肠癌中的上调的机理探讨发现Ets-1介导了INPP4B的转录上调。这些结果提示INPP4B 在结肠癌中以肿瘤驱动基因发挥功能作用，是治疗结肠癌的一个潜在靶标。.另外发现INPP4B在部分黑色素瘤中也发挥肿瘤驱动基因作用。敲低INPP4B，抑制了黑色素瘤细胞的增殖，阻滞了黑色素瘤异种移植瘤生长。反过来，过表达INPP4B，增加了黑色素瘤和色素细胞的增殖，触发了正常色素细胞的独立锚定生长。值得注意的是INPP4B介导黑色素瘤细胞增殖是通过激活SGK3，但不激活Akt。INPP4B在黑色素瘤中的上调与miR-494 a和 miR-599表达降低相关，而miR-494 a和 miR-599表达降低是则是由于基因拷贝数丢失。 在黑色素瘤细胞过表达miR-494 或者miR-599，导致INPP4B表达下调，SGK3活化降低，细胞增殖抑制。反之亦然。总之，这些结果提示INPP4B是个潜在治疗靶标，可以通过靶向INPP4B或补充恢复miR-494 和 miR-599作为新的治疗INPP4B高表达的黑色素瘤患者。

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