As the breast is a non-vital organ, primary tumor burden is very rarely the direct cause of cancer-specific mortality. Instead, metastasis of cells from the primary tumor to vital organs results in patient death. Here we demonstrate that p38γ is a novel metastasis-associated gene, which acts in part by affecting cytoskeletal orientation, cell motility, and RhoC.
The importance of the p38 MAPK pathway in cancer has been appreciated (6
), but diverse, and sometimes contradictory, roles have been described for p38 in cancer (13
). In agreement with other recent findings (15
) we show that at least some of the discrepancy in p38 function may be attributed to the distinct contributions of p38 isoforms. The p38 MAPK pathway is ubiquitously utilized in stress-response., It logically follows that metastasizing cancer cells, which encounter an ever-changing milieu of cellular stresses, may gain survival advantages under stressed conditions upon modulation of the appropriate p38 MAPK isoforms; thus, dissecting the contributions of each p38 isoform would allow more precise targeting of specific subsets of cancer. p38γ is an especially promising drug target as, in addition to the data presented here, it is a kinase, has restricted tissue expression (8
), and lacks a phenotype when knocked out in mice (29
)—possibly indicating that its inhibition may offer a differential detrimental effect on tumor versus normal cells.
Using a computational cell model in combination with live cell microscopy studies we showed that p38γ promotes BC cell motility at least in part by mediating actin cytoskeletal remodeling to create proper stress fiber orientation. Using the computational cell models, we first noted that whereas the observed whole cell locomotion could be produced by simultaneous action of all stress fibers in scrambled cells, the experimentally-observed ineffective locomotion of shp38γ cells was only possible if the action alternated between the two families of actin bundles seen in this cell phenotype—a result that would prove difficult to demonstrate using cell biological techniques alone.
The computational model predicted, and subsequent biological experiments verified, the presence of left-right leading edge protrusion oscillations in both scrambled and shp38γ cells. We note that other groups have identified oscillations in components of the leading edge (30
), however these oscillations all involved forward-backward (protrusion-retraction) movement of the leading edge as a whole. To the best of our knowledge the data presented here is thus the first evidence of left-right oscillations in the leading edge. Interestingly, the principle difference between the two cell types studied here (scrambled and shp38γ) lies in the periodicity of these oscillations. Through detailed in silico
experimentation (, Supplementary Fig. S4
) we determined that leading edge protrusion oscillation periodicity is influenced by and inseparable from cytoskeletal architecture. This inseparability sharpens the question of exactly how leading edge actin dynamics are linked to cell shape and cytoskeletal morphology. While this is a subject for future investigation, we do speculate on the chain of events: p38γ knockdown results in cytoskeletal architecture changes, which lead to alteration of cell shape, compensatory changes in leading edge protrusion periodicity, and ultimately modified whole cell motility.
Oscillation of other components of cell motility, such as cell shape (32
) and trailing edge retraction (33
), have been shown to be essential for productive cell motion in Dictyostelium
) and fish keratocytes (33
). We expect further investigations to uncover links between these processes and leading edge protrusion oscillations.
p38γ bears analogies with other mediators of normal mesenchymal differentiation such as twist and snail, which have been shown to also be important promoters of cancer progression (34
): p38γ functions in muscle cell differentiation, and thus it is consistent that it functions in mesenchymal-like BC cells. Interestingly, p38γ appears to exert its effects independently of classical cell differentiation markers, as p38γ knockdown does not alter expression of epithelial-tomesenchymal transition markers such as vimentin or E-cadherin (data not shown) despite significantly reverting the mesenchymal-like phenotype of aggressive BC cells. Although many other genes have been shown to drive metastatic transition (36
), based on the data presented here we propose that p38γ serves as a crucial regulator of the major cytoskeletal changes necessary for the switch to rapid, mesenchymal-like cell motility—independent of differentiation status—at least in part by modulating RhoC.
RhoC is involved in stress fiber formation and actomyosin contraction (37
)— both of which are perturbed by p38γ knockdown—and has previously been linked to the p38 MAPK pathway (39
). RhoC also drives metastasis in several types of cancer, including breast (37
), and plays a larger role in stress fiber formation and contraction than RhoA (43
); thus we postulated that changes in RhoC expression influence the shp38γ phenotype. Supporting this hypothesis we found that re-expressing RhoC alone was sufficient to restore motility to shp38γ cells—a surprising feat, given the multitude of proteins involved in cell motility (44
), which highlights the importance of RhoC in p38γ-mediated cell motility. Further supporting the p38γ-RhoC link is our finding that expression of the two proteins is concurrently altered in clinical BC specimens (), suggesting that the p38γ-RhoC axis may be functionally significant—and a potentially druggable target—in the clinic.
We discovered that p38γ regulates RhoC expression by preventing RhoC ubiquitination and subsequent lysosomal degradation. Although other p38 isoforms have been linked to protein ubiquitination (46
), ours is the first evidence of p38γ contributing to this process. Ubiquitination and protein degradation have recently emerged as important mechanisms for regulating Rho GTPase expression (48
), however this is the first demonstration of modulating RhoC ubiquitination as a relatively fast mechanism to regulate RhoC action within a time domain relevant to cell motion. Further research into the specific proteins and mechanisms regulating ubiquitination of RhoC and other Rho GTPases should have significant impact on our understanding of how cell motility and metastasis are regulated.