AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts
Jing Zhang, Serk In Park, Marlene C. Artime, Justin M. Summy, Ami N. Shah, Joshua A. Bomser, Andrea Dorfleutner, Daniel C. Flynn, Gary E. Gallick
Jing Zhang, Serk In Park, Marlene C. Artime, Justin M. Summy, Ami N. Shah, Joshua A. Bomser, Andrea Dorfleutner, Daniel C. Flynn, Gary E. Gallick
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Research Article Oncology

AFAP-110 is overexpressed in prostate cancer and contributes to tumorigenic growth by regulating focal contacts

Abstract

The actin filament–associated protein AFAP-110 is an actin cross-linking protein first identified as a substrate of the viral oncogene v-Src. AFAP-110 regulates actin cytoskeleton integrity but also functions as an adaptor protein that affects crosstalk between Src and PKC. Here we investigated the roles of AFAP-110 in the tumorigenic process of prostate carcinoma. Using immunohistochemistry of human tissue arrays, we found that AFAP-110 was absent or expressed at very low levels in normal prostatic epithelium and benign prostatic hyperplasia but significantly increased in prostate carcinomas. The level of AFAP-110 in carcinomas correlated with the Gleason scores. Downregulation of AFAP-110 in PC3 prostate cancer cells inhibited cell proliferation in vitro and tumorigenicity and growth in orthotopic nude mouse models. Furthermore, downmodulation of AFAP-110 resulted in decreased cell-matrix adhesion and cell migration, defective focal adhesions, and reduced integrin β1 expression. Reintroduction of avian AFAP-110 or a mutant disabling its interaction with Src restored these properties. However, expression of an AFAP-110 lacking the PKC-interacting domain failed to restore properties of parental cells. Thus, increased expression of AFAP-110 is associated with progressive stages of prostate cancer and is critical for tumorigenic growth, in part by regulating focal contacts in a PKC-dependent mechanism.

Authors

Jing Zhang, Serk In Park, Marlene C. Artime, Justin M. Summy, Ami N. Shah, Joshua A. Bomser, Andrea Dorfleutner, Daniel C. Flynn, Gary E. Gallick

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Figure 4

Effects of AFAP-110 downregulation on cell adhesion to ECM components and cell migration.

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Effects of AFAP-110 downregulation on cell adhesion to ECM components an...
(A and B) AFAP-110 downregulation–induced inhibition of cell adhesion to extracellular components. The adhesion assay was performed by seeding equal amount of cells of different groups in 96-well tissue culture plates coated with laminin (A) or type IV collagen (B). Cells were washed 3 times with PBS at 1 hour after plating, and the percentage of attached cells to total cell inputs was determined using methylthiazole tetrazolium (MTT) staining. Means and 95% CIs of the percentages of 3 independent experiments are presented; bars correspond to 95% CI. *P < 0.05, 2-tailed Student’s t test. (C and D) AFAP-110 downregulation–induced inhibition of cell migration. Migration assays were performed using Boyden chambers. Cells that migrated through 8-μm pores to the bottom side of the inserts were stained and quantified. (C) Representative images of migrated cells of different groups. Scale bars: 100 μm. (D) Numbers of migrated cells per field under ×40 magnificent microscope were quantified and expressed as means and 95% CIs of 3 independent experiments; bars correspond to 95% CI. *P < 0.001, 2-tailed Student’s t test.