In prostate cancer (PCa) the laminin binding integrin (LBI) α6β1 is involved in the extra capsular and muscle invasion of cohesive tumor clusters in part through dissemination via peripheral nerves expressing laminin. This invasion results in part due to the posttranslational modification (PTM) of the α6β1 integrin (α6) by the serine protease urokinase plasminogen activator (uPA, PLAU) and its cognate receptor (uPAR, PLAUR). The cleavage results in a tumor specific variant form of the α6 integrin called α6pβ1 (α6p). This leads to altered biophysical adhesive properties of the cohesive cancer clusters. This PTM occurs early in progression from indolent and confined tumors to aggressive and invasive phenotypes. Current strategies have the capability to detect aggressive cancers that have invaded, but are unreliable for identification of tumors that have an early signature of invasiveness. Therefore, an identification of a reliable diagnostic method that stratifies confined and indolent (low risk) tumors that lack migratory capability from one that will progress to early invasive phenotypes will aid informed and objective decisions for specific treatment strategies.
Utilizing an emerging technique of multiplex immunohistochemistry (IHC) detection of primary antibodies specific for protein biomarkers within a single formalin fixed paraffin embedded (FFPE), LBI protein interactions and associations were detected within prostate tissue samples. The interaction of LBI biomarkers with uPAR, the essential cell-cell adhesion protein E-cadherin were detected. In addition, associations with the pathway regulating tumor suppressor protein PTEN in concert with the transcription factor ERG in human prostate tissue samples were also detected. These interactions were detected in tissues exhibiting various stages of PCa disease progression. These protein interactions and associations were also the basis for generating image analysis algorithms to quantify protein expression. Using brightfield multiplex and standard DAB IHC image analysis, two separate quantitation algorithms were created and tested utilizing multiplex chromogen and IHC DAB detections. One quantitative algorithm allowed differentiation of individual chromogenic stain intensities and co-incidence of LBIs and E-cadherin biomarkers within focal regions of interest in PCa tissues. The results also displayed increased ratio of α6 integrin and E-cadherin cell-cell co-distribution in early pre-malignant events compared to aggressive tumors. The other algorithm designed identified specific localization patterns of α6 integrin in association of PTEN and ERG status. This indicated that localization of α6β1 integrin correlating to PTEN and ERG status could be used as an indicator of PCa aggressiveness.
In this study, the role of the α6β1 integrin cleavage plays in cohesive tumor invasion through muscle was characterized. A CRISPR Cas9 mouse model muscle invasion assay with DU145 prostate tumor cells injected with a transfected uncleavable α6 mutant (α6AA) exhibited significantly reduced tumor onset and extravasation (6 weeks) while mice injected with cells with a transient knockout of α3β1 integrin (α3) increased tumor burden and invasion sites in xenograft tissues. Analysis of xenograft sample tissue confirmed a significant decrease in tumor burden and reduced muscle invasion. Overall, these results suggest a loss of α3 integrin plays a role in aggressive tumor burden and muscle invasion in PCa via the cleavage of α6β1 integrin. Also, the results indicate a blockage of the α6β1 integrin cleavage demonstrate a promising mechanism to inhibit the progression of aggressive disease
