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 2

Downregulation of AFAP-110 in a prostate cancer cell line.

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Downregulation of AFAP-110 in a prostate cancer cell line. (A) AFAP-110 ...
(A) AFAP-110 protein levels in cancer cell lines LNCaP, DU145, and PC3 and a normal human prostate epithelial cell line, RWPE-1. Western blotting was performed with a monoclonal mouse anti–AFAP-110 antibody. (B) AFAP-110 expression in siRNA-expressing clones. PC3 cells were transfected with a plasmid containing an siRNA sequence targeting human AFAP-110 or a scrambled control sequence. Stable clones were established as described in Methods. Western blotting was performed with cell lysates from PC3 cells, 1 scrambled subclone, and 2 stable clones with siRNA (i.e., 309 and B11). The membranes were stripped and blotted with a monoclonal mouse anti–β-actin antibody. β-Actin expression was used as a loading control. Results from 1 of 2 independent experiments are shown. (C) Ectopic expression of GFP-tagged chicken AFAP-110. AFAP-110–downregulated clone 309 was transfected with a plasmid harboring a gene encoding a GFP-tagged chicken AFAP-110, and stable clones (309-AFAPGFP6 and 309-AFAPGFP7) were established. Western blotting was performed. β-Actin expression was used as a loading control. Results from 1 of 2 independent experiments are shown. (D) In vitro proliferation of cells as measured by counting using a hematocytometer. Parental PC3 cells (diamonds), scrambled siRNA control cells (squares), clone 309 (triangles), clone B11 (Xs), clone 309-AFAPGFP6 (asterisks), and clone 309-AFAPGFP7 (circles). Values are expressed as mean and 95% CI of experiments performed in triplicate.