High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer

Abstract

Background: 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.

Methods: Clinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed.

Results: 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.

Conclusions: 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. Video abstract.

Keywords: AMAP1; ARF6; FAK; Fibrosis; PD-L1; Pancreatic cancer.

Fig. 1

High AMAP1 expression levels statistically correlate with PD-L1 expression in human PDACs and KPC tumors. a Representative IHC images of AMAP1 with IHC scores 0 to 3 and positive and negative staining of PD-L1 in clinical specimens, and their comparison with AMAP1 IHC scores. b IHC images and quantification of the PD-L1 staining of tumors formed by control (Irr) or AMAP1-silenced (shAMAP1 #1 and #2) KPC cells in C57BL/6 mice. Error bars represent the mean ± s.e.m. **P < 0.01. c PD-L1 cell surface expression in IFNγ-treated or non-treated KPC cells, pretreated with shRNAs. MFI, median fluorescence intensity. Error bars represent the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. d Kaplan-Meier plots of the overall survival of patients with regard to PD-L1 positivity. P-values were obtained by t-tests (a, b and c) and by the log-rank test (d). Bars = 100 μm (a and b)

Fig. 2

High AMAP1 expression levels statistically correlate with fibrosis in human PDACs and KPC tumors. a Representative images of sirius red staining of clinical specimens, and Kaplan-Meier plots of overall survival of patients with regard to the staining. Bars = 200 μm. b Comparison of sirius red staining intensities in human samples with high or low AMAP1 expression or phosphorylated FAK (p-FAK) levels. c Representative images and quantification of sirius red and collagen I staining of tumors formed by control (Irr) or AMAP1-silenced (shAMAP1 #1 and #2) KPC cells in C57BL/6 mice. Error bars represent the mean ± s.e.m. Bars = 100 μm. d Representative IHC images of p-FAK and Kaplan-Meier plots with regard to the different levels of p-FAK in human PDACs. Bars = 50 μm. e Immunoblot analysis of FAK, p-FAK, and AMAP1 levels in control (Irr) and shFAK or shAMAP1 KPC cells, after culturing for 40 h in vitro. β-actin was used as a control. f A positive correlation between AMAP1 and p-FAK expression levels in human PDACs. *P < 0.05, **P < 0.01. P- values were obtained by the log-rank test (a and d) and by t-tests (b, c, and f)