Nine members of this family were diagnosed with pancreatic cancer and nine with pancreatic precancer (five with carcinoma in situ [PanIN 3] and four with low-grade dysplasia [PanIN 2]).

(A) To narrow down candidate genes we constructed custom 4q32–34 microarrays composed of 243 expressed sequence tags (IMAGE clones), each of which was spotted six times on the array. The microarrays were hybridized with RNA derived from the following target tissues: sporadic pancreatic adenocarcinoma (n = 10), Family X pancreatic precancer (n = 1), and normal donor pancreas (n = 2). Each target tissue was hybridized separately on the arrays and compared to normal pancreas. This array image is representative of one of the sporadic cancer samples versus normal pancreas. Note the consistency of the expression of the six spotted replicates of cDNA clones. Green indicates genes that were underexpressed in cancer compared to normal pancreas; red indicates genes that were overexpressed in cancer compared to normal pancreas, and yellow indicates genes that were expressed in similar levels as normal pancreas.
(B) The most overexpressed clones among ten sporadic pancreatic cancers were two different palladin clones; both of these clones were significantly overexpressed in Family X precancer.
(C) RNA overexpression of palladin was confirmed by qRT-PCR. Samples tested were whole pancreatic tissues and included six new normal pancreas samples, four new precancerous tissues (PanIN 2 and 3, or low- and high-grade dysplasia, respectively) from two Family X individuals and two unique familial pancreatic cancer individuals not from Family X, nine histologically normal tissues adjacent to cancer, and 16 new pancreatic cancer tissues. In each case, palladin expression was measured relative to the endogenous standardized control, GAPDH. Error bars indicate 1 standard deviation above and below the average. Palladin RNA is overexpressed early in the process of pancreatic neoplasia, as it is overexpressed in normal-appearing tissue adjacent to sporadic cancer, as well as in precancerous pancreatic tissue. In addition, palladin is overexpressed in the precancerous tissue from three different familial pancreatic cancer kindreds (Family X and two other familial pancreatic cancer kindreds).

Top black bar indicates the genomic location of the Family X mutation between microsatellite markers D4S413 and D4S299 on Chromosome 4. The center line designates the location of the palladin transcript, AB023209, with vertical boxes and lines indicating exons. At the bottom, a small portion of the cDNA sequence shows a C to T base pair change at position 715 (indicated with an arrow), which causes a proline (hydrophobic) to serine (hydrophilic) amino acid change at amino acid 239 (P239S). This location is the essential binding site for alpha-actinin, another key cytoskeletal protein.

The mutation in Family X causes a proline (hydrophobic) to serine (hydrophilic) change in the human amino acid sequence.

Palladin binds other key proteins such as Ezrin [16]. Previous studies have shown that Ezrin and S100P are proteins that are abnormally regulated in pancreatic cancer [35,36].

Expression of Palladin RNA increases relative to the degree of precancer to cancer: normal to PanIN 1 (hyperplasia) to PanIN 3 (carcinoma in situ) to cancer. Each bar represents epithelial cell cultures from one person. The PanIN 1 and PanIN 3 lesions (lower left photomicrograph) were purified from two affected members of Family X. The pancreatic cancer epithelial cells (lower right photomicrograph) were purified from a sample of sporadic adenocarcinoma.

Proteins were detected using polyclonal antibody to palladin raised in rabbit (ab 621) in this Western blot. The major palladin isoform expressed in normal HPDE and cultured epithelial cells is the 90 kDa isoform (solid arrows). The positive control sample (MSC, human mesenchymal stem cells) also has the major 90 kDa isoform, but the 140 kDa isoform is also present (dashed arrow). Mesenchymal stem cells express high levels of palladin.

Human mesenchymal cell lysate (hMCL) was used as a positive control for palladin overexpression. FA6, HPAF, IMIMPC2, SUIT2, and PATU2 are sporadic pancreatic cancer cell lines that overexpress palladin protein compared to the minimal expression evident in normal HPDE. Alpha-actinin is also abnormally expressed in some of the pancreatic cancer cell lines compared to normal pancreas—especially in PANC-1, one of the few pancreatic cancer lines that does not show expression changes in palladin protein.

(A and B) When HeLa cells were transfected with the human wild-type (WT) palladin construct, filamentous bundles (A) clearly colocalized with the F-actin (B).
(C and D) In contrast, expression of the mutant P239S Family X (FX) construct resulted in large palladin flower-shaped structures (arrows) in which the center, but not the petals of the flower, colocalizes with actin.
(E and F) Expression of Family X constructs can also cause small cytoplasmic flecks of palladin that do not colocalize with actin.
HeLa cells overexpressing the empty GFP vector displayed uniform distribution of GFP in the whole cells that did not colocalize with F-actin (unpublished data). Green, GFP-palladin; red, F-actin; blue, nucleus; yellow, colocalized palladin and F-actin. All figures are at 100× magnification.

The cells expressing the Family X palladin construct (P239S) migrated fastest—33% and 42% faster than the cells expressing wild-type (WT) protein and empty vector constructs, respectively.