AFAP1L1, a novel associating partner with vinculin, modulates cellular morphology and motility, and promotes the progression of colorectal cancers

Abstract

We have previously identified actin filament-associated protein 1-like 1 (AFAP1L1) as a metastasis-predicting marker for spindle cell sarcomas by gene expression profiling, and demonstrated that AFAP1L1 is involved in the cell invasion process by in vitro analyses. However, its precise molecular function has not been fully elucidated, and it remains unknown whether AFAP1L1 could be a prognostic marker and/or therapeutic target of other malignancies. In this study, we found a marked elevation of AFAP1L1 gene expression in colorectal cancer (CRC) tissues as compared to the adjacent normal mucosa. Multivariate analysis revealed that AFAP1L1 was an independent and significant factor for the recurrence of rectal cancers. Moreover, the addition of the AFAP1L1 expression level to the lymph node metastasis status provided more predictive information regarding postoperative recurrence in rectal cancers. AFAP1L1-transduced CRC cells exhibited a rounded shape, increased cell motility on planar substrates, and resistance to anoikis in vitro. AFAP1L1 localized to the ringed structure of the invadopodia, together with vinculin, and AFAP1L1 was identified as a novel associating partner of vinculin by immunoprecipitation assay. AFAP1L1-transduced cells showed accelerated tumor growth in vivo, presumably reflecting the anoikis resistance of these AFAP1L1-expressing cells. Furthermore, the local administration of a siRNA against AFAP1L1 significantly suppressed the in vivo tumor growth of xenografts, suggesting that AFAP1L1 might be a candidate therapeutic target for CRCs. These results suggest that AFAP1L1 plays a role in the progression of CRCs by modulating cell shape and motility and by inhibiting anoikis, presumably through interactions with vinculin-including protein complexes.

Regulation of sarcoma cell migration, invasion and invadopodia formation by AFAP1L1 through a phosphotyrosine-dependent pathway

“…These results provide an important mechanistic explanation for the regulation of invadopodia by AFAP1L1 in sarcoma cells. Our novel findings also significantly expand the recent findings on AFAP1L1 interacting with vinculin at invadopodia and its regulation of disease progression in colorectal cancers, 26 and provide an impetus to delineate whether the Vav2/Nck2 connection to AFAP1L1 also mediates its links to colorectal cancer progression.…”

“…AFAP1L1 could also be directly interacting with Tks5 and p190RhoGAP within invadopodia via their SH3 domains through binding to PXP motifs within AFAP1L1, similar to that reported for AFAP1. 27 These observations together with the ability of AFAP1L1 to directly bind the invadopodia/podosome marker cortactin 8 , associate with vinculin (found at focal adhesions as well as invadopodia) 26 and the fact that AFAP1L1 encompasses a homologous site to that which is phosphorylated by protein kinase C in AFAP1 (which regulates podosome and potentially invadopodia lifespan), 11 we now have significant evidence for placing AFAP1L1 as a crucial regulator of invadpodia in sarcoma cells, and that it directly intersects multiple critical invadopodia regulatory networks. The strongest evidence for the importance of AFAP1L1 for invadopodia regulation, from other investigators 8,9,26 and as we report here, relates to the U2OS cell line.…”

“…1,[45][46][47] In addition, our data show that signaling from AFAP1L1 is modulated by a cell's contact with the extracellular matrix, which could explain the differential effects on cell growth in twodimensional monolayer cultures (which show no major effects on cell proliferation) compared with three-dimensional and in vivo cultures (which show significant effects on proliferation/survival) of cells over/underexpressing AFAP1L1. 8,26 Although high expression of total AFAP1L1 is associated with sarcomas (and colorectal cancer progression), 8,26 it will be important to determine whether the activation status (that is, phosphorylation of Y136 and Y566 sites, for which we have generated specific antibodies) also correlate with disease status and clinical outcomes. Important clinical applications of the molecular understanding we now have of the AFAP1L1 pathway need to be addressed through the use of both advanced cancer mouse models with altered AFAP1L1 (for example, sarcomas bearing wild-type or Y136/566F mutant AFAP1L1) and identifying therapeutic avenues to disrupt the AFAP1L1 pathway in sarcoma and potentially other (for example, colorectal cancer) patients.…”

Cytomorphological Characterization of Individual Metastatic Tumor Cells from Gastrointestinal Cancer Patient Lymph Nodes with Imaging Flow Cytometry

“…The current study demonstrates that IFC is an ideal technology to carry out such studies. IFC may also prove useful to evaluate expression of additional markers (e.g., stem cell CD133, CD44 [38], mesenchymal, or other markers including AFAP1L1 [41]) which in combination with individual morphological characterization could potentially allow for the selection of personalized treatments. This technique could also be applied to the analysis of DTC in bone marrow for which morphological and phenotypical analysis could be extremely advantageous for improving risk prevention and tailored treatment [42].…”

Peptidomic Analysis via One-Step Direct Transfer Technology for Colorectal Cancer Biomarker Discovery

“…The following references discuss the various candidate peptides in more detail: alpha-1-antichymotrypsin [34], alpha-1-antitrypsin [35][36][37][38], alpha-2-HS-glycoprotein [20,[39][40][41][42], F. XIIIa [43], fibrinogen alpha chain [21], PF4 [21], prothrombin [43], vasodilator-stimulated phosphoprotein [44][45][46], and vinculin [47][48][49][50]. However, altered expression of these factors has not been associated with CRC and their known functions are not consistent with CRC pathology.…”