Characterization of Leukocyte Formin FMNL1 Expression in Human Tissues

Abstract

Formins are cytoskeleton regulating proteins characterized by a common FH2 structural domain. As key players in the assembly of actin filaments, formins direct dynamic cytoskeletal processes that influence cell shape, movement and adhesion. The large number of formin genes, fifteen in the human, suggests distinct tasks and expression patterns for individual family members, in addition to overlapping functions. Several formins have been associated with invasive cell properties in experimental models, linking them to cancer biology. One example is FMNL1, which is considered to be a leukocyte formin and is known to be overexpressed in lymphomas. Studies on FMNL1 and many other formins have been hampered by a lack of research tools, especially antibodies suitable for staining paraffin-embedded formalin-fixed tissues. Here we characterize, using bioinformatics tools and a validated antibody, the expression pattern of FMNL1 in human tissues and study its subcellular distribution. Our results indicate that FMNL1 expression is not restricted to hematopoietic tissues and that neoexpression of FMNL1 can be seen in epithelial cancer.

Keywords: FMNL1; FRL1; actin cytoskeleton; formin; immunohistochemistry.

Figure 1.

The domain structure of FMNL1 isoforms and the regions used for antibody production and transfections. FMNL1 isoforms 1 and 2 show only minor variations in their C-terminal region. The C-terminus of isoform 3 differs from the others because of an intron retention, which partly changes the DAD domain and endows constitutive activation of this isoform. The antigenic sequence for antibody production is within the common FH2 domain. The construct used for transfections harbors most of the FH2 domain and the DAD domain of isoform 1.

Figure 2.

Detection of the FMNL1’ construct and endogenous FMNL1 by western blotting and fluorescence microscopy. (A) HEK 293T cells were transfected with pCMVsport6-FMNL1’, pEGFP-FMNL1’ or empty pEGFP expression constructs. In control cells transfected with empty pEGFP, no FMNL1 band was detected in western blotting. In transfected cells, a 130-kDa band corresponding to GFP-tagged FMNL1’, and a 100-kDa band corresponding to non-tagged FMNL1’ is visible. (B) Endogenous FMNL1 is detected as a single band of 140 kDa in several cell lines: myofibroblasts (CCD18), T-cell lymphoma (Jurkat), breast cancer (MDA-MB-231) and ovarian cancer (Ovcar-4 and Hey). (C) To confirm that the FMNL1 antibody does not cross-react with FMNL2 or FMNL3, cell lysates were first blotted with the FMNL1 antibody, and reprobed with an antibody that cross-reacts with both FMNL2 and FMNL3. In MDA-MB-231 cells, a single band corresponding to FMNL1 was visible in MDA-MB-231 cells but not in SK-Mel-28 melanoma cells. In both cell lines, bands corresponding to FMNL2 and FMNL3 were present, indicating that the FMNL1 antibody specifically detects FMNL1, not FMNL2 or FMNL3. The specificity of the FMNL2 and FMNL3 antibody was further confirmed by reduced reactivity after corresponding siRNA treatments. Tubulin was blotted as a loading control. (D) IGROV1 ovarian cancer cells and the WM164 and SK-Mel-28 melanoma cell lines, which do not express endogenous FMNL1, were transfected with an FMNL1’ construct and stained with green phalloidin to visualize actin filaments (upper panel) and FMNL1 (red, middle panel). In membrane protrusions, FMNL1’ co-localizes with bundled F-actin (merge in yellow, bottom panel). Higher magnification of membrane protrusions are depicted in insets. (E) IGROV1, WM164, and SK-Mel-28 cells were transfected with a construct expressing GFP-FMNL1’ or GFP only. GFP shows a diffuse cytoplasmic distribution (upper panel), whereas GFP-tagged FMNL1’ is partly located at the membrane protrusions. Scale bars, 20 μm. Nuclei were stained with DAPI (blue).

Figure 3.

FMNL1 mRNA expression profile in human normal and neoplastic tissues. The normalized expression values (y-axis) of FMNL1 across normal tissues and cancer types are presented as box-plots. The box extends from the first to the third quartile of the data and the median is indicated as a green line for normal tissue, and a red line for cancer. The whiskers extend to the extreme values unless there are outliers. The data observations that lie more than 1.5 × interquartile range (IQR) lower than the first quartile or 1.5 × IQR higher than the third quartile were considered as outliers and are indicated separately. The highest expression levels are seen in bone marrow myeloid cell and lymphatic system and their malignancies. In most tissues and cancers, FMNL1 expression is low or absent. Among cancer tissues, a substantial number of cases express FMNL1 more than the corresponding normal tissues.

Figure 4.

Distribution of FMNL1 in human tissues. (A) In the bone marrow, megakaryocytes and erythroid cells do not express FMNL1, whereas myeloid cells stain strongly (arrow). (B) In lymph nodes, mature lymphocytes stain strongly. (C) In the colon, epithelial cells do not express FMNL1, whereas stromal lymphocytes and smooth muscle show strong reactivity (arrowhead). (D) Prostatic epithelium does not express FMNL1, whereas stromal smooth muscle cells stain strongly (arrowhead). (E) In the placenta, trophoblastic cells express FMNL1. (F) Skeletal muscle cells stain weakly. (G) In the female breast, epithelial cells express little FMNL1. Myoepithelial cells, however, stain strongly (arrowhead). (H) The central nervous system is an example of tissues where parenchymal cells do not express FMNL1. Neither neurons nor glial cells express FMNL1. (I) In the uterus, endometrial epithelial cells and endometrial stromal cells do not express FMNL1. Small lymphocytes stain positively in the endometrial stroma. Myometrial (smooth muscle) cells stain strongly (arrowhead). Detailed staining is presented in insets. Scale bar, 200 μm.

Figure 5.

FMNL1 staining in basal type breast cancer cells and tissues. (A) MDA-MB-231 breast cancer cells stained for F-actin and FMNL1. FMNL1 is present in lamellipodia, at filopodia tips (inset), and in the cytoplasm. Scale bar, 20 μm. (B) Among basal type breast cancers, FMNL1 expression is heterogeneous. In the left micrograph, cancer cells across the tumor are virtually negative, whereas strong staining is seen in lymphocytes and in other inflammatory cells present in the tumor (arrowhead). In the middle and right micrographs, most breast cancer cells express little FMNL1. However, subpopulations of cells near the tumor margin show strong reactivity (arrows). Scale bar, 200 μm. (C) FMNL1 (brown) and CD68 (red) double staining of basal breast cancers demonstrates that part of the FMNL1-positive cells are CD68-negative carcinoma cells and therefore distinct from macrophages. Scale bar, 200 μm.