A. Rajput1, G. Wan1, A. Rajput1 1University Of New Mexico HSC,Surgical Oncology/Surgery,Albuquerque, NM, USA
Introduction:
Phosphatidylinositol 3-kinase (PI3K) is a lipid kinase through which a number of receptor tyrosine kinases signal. Gain of function mutations in the catalytic subunit p110α (PIK3CA) of Class 1A PI3K occur in up to one-third of human colorectal cancers (CRC), and result in dysregulation of cell signaling. Our group has previously shown that in in vivo orthotopic models, that human colon cancer cell lines bearing the H1047R point mutation in p110α are more metastatic than cells carrying wild type p110α. The purpose of this study was to compare changes in cell morphology in wild type and mutant PI3K bearing cells. We hypothesized that the H1047R mutation in PI3K would result in rearrangement of the cytoskeleton and thus morphology and cell motility.
Methods:
1) HCT116 cells with either the WT or the MUT PIK3CA allele alone were grown and fixed on coverslips. Following permeablization, F-actin was labeled by AlexaFluorR 488 phalloidin and confocal images of F-actin labeled WT and MUT cells were acquired. The cell morphology was analyzed by Image J and the degree of difference between the structures of actin filopodia in WT and MUT cells was assessed using a custom Matlab script. The actual length of the cell border between the start and end points of these line segments was then measured. The ratio between the sum of the lengths of the 5µm line segments and the total cell border length, R (eq 1), is a measure of the surface roughness of the cell, which corresponds to the number of filopodia present. Therefore, an R-value close to 1 indicates a smooth surface, whereas an R-value close to 0 is a rougher surface. 2) F-actin level was measured by Flow Cytometer and 3) cell motility was tested by wound healing assay.
Results:
HCT116 H1047R MUT bearing cells compared to WT PIK3CA cells demonstrated increased levels of PIP3 which is reflective of gain of PI3K function. There was also a decrease in F-actin levels in MUT vs. WT bearing cells. MUT bearing cells also demonstrated significantly increased numbers of cell filopodia as shown in Figure 1. Wound healing assay demonstrated increased cell motility in MUT as compared to WT cells.
Conclusion:
Our findings indicate that the H1047R mutation reorganizes actin structure; generates higher PIP3 levels and decreases F-actin levels. This function possibly contributes to the enhanced migratory capacity of HCT116 MUT cells. Our results further confirmed that the accumulation of PIP3 and change in the appearance of cytoskeleton of cells are important aspects in regulating cell motility and therefore possibly metastasis. Thus the PI3K pathway remains a desirable therapeutic target for patients with colorectal cancer.
