Not merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis. Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed. Elevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients. Together with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK.

中文翻译：

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ARF6 效应子 AMAP1 的高表达与 PD-L1 水平升高和胰腺癌纤维化有关。

不仅仅是免疫逃避的开始，还有其他因素，如酸中毒和纤维化，也是癌症治疗的主要障碍。致密纤维化是胰腺导管癌 (PDAC) 的标志，其中肿瘤细胞中粘着斑激酶 (FAK) 的过度活化被证明是至关重要的。KRAS/TP53 双突变是 PDAC 的特征。我们之前表明，ARF6 及其下游效应子 AMAP1 的高蛋白表达，以及细胞表面酪氨酸激酶受体激活 ARF6 的过程，是 KRAS/TP53 突变促进 PDAC 侵袭、转移和免疫逃避的主要靶点。 . 这一观点被人类 PDAC 的 KPC 小鼠模型（LSL-Kras（G12D/+）；LSL-Trp53（R172H/+））重新捕获；Pdx-1-Cre)。从机制上讲，ARF6-AMAP1 通路主要参与 PD-L1 的细胞动力学，β1-整联蛋白和 E-钙粘蛋白；从而在 ARF6 被激活时调节细胞粘附特性。在这里，为了了解 ARF6-AMAP1 通路是否与 PD-L1 水平升高和 PDAC 纤维化有关，我们分析了 AMAP1 与这些恶性表型之间的关系。此外，由于 ARF6 通路可能与粘着斑动力学密切相关，因此与 FAK 密切相关，我们还研究了 AMAP1 是否在纤维化中使用 FAK。分析了临床标本以及 KPC 细胞/肿瘤及其 shAMAP1 或 shFAK 衍生物。PD-L1 水平升高和纤维化与我们患者队列的不良结果相关，与之前的报告一致；其中高 AMAP1 表达与升高的 PD-L1 和纤维化具有统计学相关性。为了保持一致，KPC 细胞中 AMAP1 (shAMAP1) 的沉默导致其肿瘤中 PD-L1 表达降低和纤维化。另一方面，shAMAP1 仅轻微影响 KPC 细胞中的 FAK 活化，磷酸化的 FAK 与增强的纤维化或我们患者的不良预后无关。连同我们之前的数据，我们的结果共同表明，由 KRAS/TP53 突变赋予的 ARF6-AMAP1 通路与 PD-L1 表达升高和人 PDAC 纤维化密切相关，将在 KPC 小鼠模型中重新捕获。ARF6 通路可能会促进纤维化，而与 FAK 无关。连同我们之前的数据，我们的结果共同表明，由 KRAS/TP53 突变赋予的 ARF6-AMAP1 通路与 PD-L1 表达升高和人 PDAC 纤维化密切相关，将在 KPC 小鼠模型中重新捕获。ARF6 通路可能会促进纤维化，而与 FAK 无关。连同我们之前的数据，我们的结果共同表明，由 KRAS/TP53 突变赋予的 ARF6-AMAP1 通路与 PD-L1 表达升高和人 PDAC 纤维化密切相关，将在 KPC 小鼠模型中重新捕获。ARF6 通路可能会促进纤维化，而与 FAK 无关。