The data presented here identify palladin as the mutated gene in the pancreatic cancer susceptibility locus at 4q32–34, validate abnormal expression of the gene in a familial pancreatic cancer kindred, demonstrate RNA overexpression of this gene in sporadic pancreatic cancers and precancerous pancreatic tissues, characterize the functional changes induced by the mutant protein, and suggest that cytoskeletal abnormalities may be a driving force in pancreatic oncogenesis.
is named for a Renaissance architect, Palladio, because of its role as a key architectural element of the cell. The function of palladin is to provide a scaffold for cytoskeletal proteins to bind, forming the actin filament complexes necessary for cell morphology, movement, and differentiation [16
]. Abnormalities in the proteins that arrange the cytoskeleton can result in loss of cellular polarity (as the cytoskeleton helps direct the location and size of the nucleus in the cell), changes in cell size, increased invasiveness (advancement and retraction of the lamellipodia moving the cell), and abnormal signaling within the cell (the cytoskeleton scaffold is the docking point for many protein-protein interactions).
The cytoskeleton is composed of actin bundles (polymerized filaments) that form the “tent poles” supporting the cell membrane. The tented cell is held onto the basement membrane by integrin pegs, with the cytoskeleton directly connected to the pegs. It is through this normal integrin-cytoskeleton interaction that the cell is able to distinguish down from up, providing normal polarity for the cell and a stationary position [27
]. During cell migration, the cross-linked network of actin filaments is dynamic, forming and dissolving through regulation by specialized actin-binding proteins; in essence these actin-binding proteins form complexes and a scaffold for the actin to polymerize. Disruption of the cytoskeleton has been directly linked to metastatic potential of cancer cells [28
]. Specifically, highly metastatic cells, which have poor cytoskeletal architecture, can detach easily from the primary tumor mass, form transient and weak connections with surrounding connective tissue, and rapidly migrate through it [29
To make the discovery of mutated palladin as a cause of familial pancreatic cancer possible, we had to devise an endoscopic surveillance program that could detect the precancerous lesions of the pancreas. This program, to our knowledge the first of its kind, allowed the identification of affected members in Family X, a large kindred with autosomal dominant, highly penetrant pancreatic cancer. Genotyping linked the pancreatic susceptibility gene to 4q32–34. A custom microarray of the dense 4q32–34 region led to the detection of palladin overexpression in Family X precancer and in sporadic cancer as well. RNA overexpression of palladin was validated by qRT-PCR in whole tissue of four precancerous samples (two from members of Family X and two from non-Family X patients) and in 16 sporadic cancers. Palladin was also overexpressed in the apparently “normal” tissue adjacent to the cancer. Specific analysis of the ductal epithelium revealed an apparent dose-response of palladin levels in the pancreatic epithelial cells, with increasing RNA expression directly associated with increasing neoplastic degeneration. These findings suggest that palladin plays a very early role in pancreatic tumorigenesis in both the familial and sporadic forms of the disease and that malignant progression parallels increased expression of the gene.
Once the candidate gene was identified, sequencing verified that a mutation in the cytoskeleton scaffold gene palladin causes familial pancreatic cancer in Family X. We demonstrated that the mutated allele is expressed and that the consequence of the P239S mutation is a change in amino acids at the essential alpha-actinin binding site leading to distinctive changes in the actin bundle morphology. Cells expressing the P239S mutant palladin protein were found to be significantly more mobile than those expressing wild-type palladin—theoretically, this capability would provide an advantage for cells to invade surrounding structures (a signature feature of cancer).
Palladin is a key scaffold for the cytoskeleton—it binds several proteins including alpha-actinin [20
]. The palladin
mutation in Family X leads to an amino-acid change in the highly conserved and essential binding site for alpha-actinin () [20
]. We were able to detect protein expression abnormalities in either palladin and/or alpha-actinin in six of seven of the sporadic pancreatic cancer cell lines that we tested. This change in the palladin/alpha-actinin axis suggests that these proteins are essential for intact normal cellular behavior. Specifically, the alpha-actinin/palladin protein axis establishes a link between B-integrin subunits and filamentous actin, holding the cell (through the cytoskeleton) to the basement membrane (unpublished data) [16
]. Through this action, normal pancreatic epithelial cells are uniform in size, oriented to the basement membrane, and immobile—exactly the cellular features that are lost in cancer. Alpha-actinin also plays a role in formation of complex/adhesion plaques; these plaques are modulated during growth and differentiation (and reduced in tumor cells) [29
]. Mutations in the alpha-actinin binding site of palladin have not previously been recognized in human disease, although decreased levels of normal alpha-actinin in 3T3 cells cause tumorigenesis in nude mice [30
Complete loss of palladin function in knock-out mice results in embryonic lethality [31
]. It is apparent that at least some palladin protein must be present for the cell differentiation and migration events that occur during normal development. Mutation, rather than complete loss, may lead to partially intact but imperfect cytoskeleton scaffold function. In support of this hypothesis, the Family X mutated constructs revealed that actin cytoskeleton bundles can still form, although they are aberrant.
It appears likely that palladin
is a proto-oncogene. The protein is overexpressed in sporadic pancreatic cancer, and even in the normal-appearing tissue adjacent to the cancer. The molecular events that cause this overexpression are unknown. Copy number in the palladin
region (4q32–34) has not been reported to be abnormal (either gains or losses) in previous comparative genomic hybridization analyses [22
]. The copy number analysis that we performed in the precancerous tissue of Family X members revealed two copies of the gene in every sample tested. These findings make it less likely that palladin overexpression is achieved through amplification. Rather, palladin levels might be elevated through point mutations, such as the one in Family X, or through other mechanisms, such as promoter region changes, epigenetic changes, or regulatory factor alterations. It is also likely that palladin does not act alone. The vast majority of pancreatic cancers have activation of the K-ras
oncogene, including in Family X [7
]. The idea that palladin
may work together is an interesting conjecture. Both of the encoded proteins regulate normal cytoskeletal activity, and the joint overexpression and activation of these genes might induce inescapable failure of normal cytoskeleton function.
Pancreatic cancer biology has been difficult to probe. Although genetic defects associated with pancreatic cancer have been described, a cohesive explanation of pancreatic neoplastic progression is lacking. The concept that the cytoskeleton could play a key role in pancreatic cancer formation is compelling. The critical features that underlie cancer, including nuclear atypia, loss of cellular polarity, altered cell morphology, increased mobility, and invasion into surrounding structures, are all linked to the dynamic behavior of the cytoskeleton. Moreover, altered cytoskeletal tension can distort the attached nuclear envelope, rearranging chromatin/gene positions, subjecting DNA to increased mutations, and inducing resistance to chemotherapeutic agents [32
Our data support a key role for palladin in the formation of sporadic pancreatic cancer and precancer; moreover an inherited mutation in the palladin gene leads to a highly penetrant autosomal dominant form of familial pancreatic cancer. However, the work described is limited to one familial pancreatic cancer kindred—it will be interesting to evaluate additional familial pancreatic cancer kindreds for mutational changes in palladin. The work is also limited in terms of understanding the role of palladin in sporadic pancreatic cancer. It seems unlikely that the P239S germline mutation will be a useful tool in genetic testing for pancreatic cancer risk in individuals without a strong family history of pancreatic cancer.
Many questions remain. Do somatic palladin mutations play a role in pancreatic cancer? How is the gene activated in the sporadic setting? Are other binding partners of palladin abnormally expressed or mutated in pancreatic cancer (sporadic or familial)? How does palladin interact with the products of oncogenes and tumor suppressor genes in known pancreatic cancer pathways? Answers to these questions are crucial to improved methods of early detection and develop new treatments for this devastating disease.