The Axl receptor tyrosine kinase confers an adverse prognostic influence in pancreatic cancer and represents a new therapeutic target

Abstract

Background: Pancreatic cancer is a near uniformly lethal disease and a better understanding of the molecular basis of this malignancy may lead to improved therapeutics. The Axl receptor tyrosine kinase is implicated in cellular transformation and tumor progression, although its role in pancreatic cancer has not been previously documented.

Results: Axl labeling was present in 54 of 99 (55%), and was absent in 45 of 99 (45%) cases, respectively. Axl expression in pancreatic cancer was significantly associated with lymph node metastases (p < 0.01), and a shorter median survival (12 versus 18 months, p < 0.01), than in tumors with negative labeling. Stable knockdown of Axl resulted in significant reduction in cell viability (p < 0.001), anchorage independent growth (p = 0.0031), as well as attenuation of migratory (p < 0.001) and invasive properties (p < 0.005), compared to vector-transfected cells. Profound inhibition of p42/p44 MAP kinase and PI-3kinase/Akt effector pathways was observed in MIAPaCa-2 cells with loss of Axl function. The reduction in invasion and migration upon Axl knockdown was mirrored by a decrease in the amounts of activated (GTP-bound) GTPase proteins Rho and Rac, significant downregulation in transcript levels of the epithelial mesenchymal transition (EMT)-associated transcription factors slug, snail and twist, and significant decrease in matrix metalloproteinase MMP-9 mRNA levels.

Materials: The immunohistochemical expression of Axl protein was assessed in a panel of 99 archival pancreatic cancers. Endogenous Axl expression was stably downregulated by lentiviral short hairpin shRNA directed against AXL mRNA in MIAPaCa-2 cells, and the effects on cell viability, anchorage independent growth, invasion, migration and intracellular effector pathways was assessed, by comparing to lentiviral vector-transfected cells.

Conclusion: Expression of Axl tyrosine kinase in pancreatic cancers confers an adverse prognostic influence, and represents a new therapeutic target in this malignancy.

Figure 1

The Axl receptor tyrosine kinase is overexpressed in pancreatic cancer. (A) A pancreatic cancer with absence of Axl labeling. Only the intra-tumoral blood vessels are positive for Axl expression. (B) A pancreatic cancer with diffuse overexpression of Axl in the neoplastic cells. Note absence of labelling in adjacent normal ductal epithelium (arrow). (C) Higher magnification of a pancreatic cancer with Axl expression. The infiltrating adenocarcinoma shows cytoplasmic staining with membranous accentuation for Axl, consistent with the pattern of expression for a receptor tyrosine kinase. (D) Axl expression is associated with adverse prognosis in pancreatic cancer. Kaplan Meier survival analysis confirms that patients with Axl-expressing tumors demonstrate a significantly shorter median survival than those with Axl-negative cancers (12 months versus 18 months, p < 0.05).

Figure 2

Axl expression in pancreatic cancer cell lines and stable knockdown of Axl in MIAPaCa-2 cells. (A) Axl protein is overexpressed in three of five pancreatic cancer cell lines (PANC-1, CFPAC1 and MIAPaCa-2), compared to the hTERT immortalized human pancreatic epithelial line HPNE. (B) Endogenous Axl was downregulated in the Axl-expressing MIAPaCa-2 cells using a lentiviral shRNA vector. Compared to vector-transfected (“mock”) clones, the shRNA-expressing cells demonstrate essentially complete loss of Axl protein. (C) Quantitative real-time PCR (qRT-PCR) confirms the downregulation of AXL transcripts in shRNA expressing MIAPaCa-2 clones. The assays were performed in triplicate and SDHA was used as housekeeping control.

Figure 3

Knockdown of endogenous Axl in MIAPaCa-2 inhibits in vitro cell viability and anchorage independent growth. (A) In vitro cell viability of Axl shRNA-expressing MIAPaCa-2 cells was significantly reduced compared to vector-transfected cells (p < 0.0001), as measured using MTS assay. The MTS assays were performed in triplicate, and mean and standard deviations are plotted. (B) Anchorage independent growth of Axl shRNA-expressing MIAPACA-2 cells, as assessed by colony formation in soft agar, was significantly reduced compared to vector-transfected cells (p = 0.0031). Colony assays were performed in triplicate, and the mean and standard deviations of colony counts were calculated for each condition. (C) Representative soft agar assay of Axl shRNA-expressing MIAPACA-2 compared to vector-transfected cells.

Figure 4

Knockdown of endogenous Axl in MIAPaCa-2 cells inhibits in vitro invasion and migration. (A) Modified Boyden chamber assay (with Matrigel plug) was performed to assess in vitro invasion in MIAPaCa-2 cells. At 72 hours, loss of endogenous Axl function was associated with significant reduction in invasive capacity compared to vector-transfected cells (p < 0.0005), when normalized for cell viability. The histogram represents mean and standard deviation of invasion assay performed in triplicate. (B) Modified Boyden chamber assay (without Matrigel plug) was performed to assess in vitro migration in MIAPaCa-2 cells. At 72 hours, loss of endogenous Axl function was associated with significant reduction in migratory capacity compared to vector-transfected cells (p < 0.0001), when normalized for cell viability. The histogram represents mean and standard deviation of migration assay performed in triplicate.

Figure 5

Knockdown of endogenous Axl is associated with reduction in filopdial extensions and loss of polarity in MIAPaCa-2 cells. Immunofluorescence studies demonstrate that vector-transfected MIAPaCa-2 cells have a spindled morphology, with well formed filopdial extensions as seen by β-tubulin/actin compound immunostaining. In contrast, loss of Axl is associated with loss of polarity and cell rounding, and reduction in the filopodial extensions. DAPI is used as a nuclear counterstain.

Figure 6

Multiple intracellular effector pathways are disrupted upon inhibition of endogenous Axl function in MIAPaCa-2 cells. (A) Lentiviral shRNA-mediated stable knockdown of Axl in MIAPaCa-2 cells blocks the activation of critical intracellular effector pathways known to be activated in pancreatic cancer, including the MAP kinase and PI-3-kinase/Akt signaling pathways. Western blot assay was performed for phospho-ERK1/2, phospho-Akt^ser473, and phospho-PDK1^ser241, all of which demonstrated reduction in levels of specific phosphorylated protein, compared to vector-transfected cells. In contrast, no changes were seen in the levels of total Akt protein. Actin was used as loading control. (B) Knockdown of endogenous Axl is associated with reduction in GTP-bound (active) Rho and Rac in MIAPaCa-2 cells compared to vector-transfected cells. The immunoprecipitation assay specifically “pulls down” GTP-bound forms of both proteins. (C) qRT-PCR demonstrates that knockdown of endogenous Axl results in significant reduction in transcripts for snail (p = 0.0004), slug (p = 0.005), and twist (p = 0.0254), whose protein products are transcription factors implicated in epithelial-mesenchymal-transition (EMT), compared to vector-transfected MIAPaCa-2 cells. Loss of Axl function was also associated with significant reduction in transcripts for the matrix metalloproteinase MMP-9 (p = 0.0004). All qRT-PCR assays were performed in triplicate, and GUSB was used as housekeeping control. The Y-axis represents relative fold level in Axl shRNA-expressing cells normalized to vector-transfected cells, and mean and standard deviations are plotted.