PDCD4 shows promise as a biomarker, with prognostic attributes in a number of cancers (4
). Here, we further find that PDCD4
expression is informative with regard to survival of breast cancer patients, where increased levels correlate with better outcome in two large scale clinical evaluations. PDCD4
expression alone, however, has limitations in stratifying cancer patient outcomes as ~30% of patients with tumors expressing relatively high PDCD4
levels have poor survival. This could be due to a potential discrepancy between mRNA and protein levels (26
), to environmental factors, or to interacting regulatory pathways.
The discovery here of PRMT5 as an interacting partner opened a new level of regulation to consider. Neither PDCD4 nor PRMT5 affected tumor cell growth when expressed alone in our model system. Although this was somewhat surprising, it may indicate that in a particularly aggressive or advanced tumor context, elevated expression of either protein alone is insufficient to alter tumor properties. In the case of PDCD4, this may be due at least in part to endogenous levels of PRMT5. Importantly, the orthotopic tumor model revealed that, together, elevated PDCD4 and PRMT5 expression significantly enhanced tumor growth. This result suggests that PRMT5 does not just negate a PDCD4 tumor suppressor function, but works synergistically to promote tumor growth. This pro-growth effect occurred only in the tumor context, not in tissue culture, hinting that the combination of proteins activates a pathway that enhances the ability to establish a productive tumor microenvironment. The similar numbers of cells staining for activated caspase-3 and Ki67 in tumors expressing elevated levels of both PDCD4 and PRMT5 versus tumors with vector alone suggests that alteration in apoptosis or proliferation does not account for the larger tumor size in the former case. It will be important to investigate these features at earlier points in tumorigenesis. Changes in early events coordinated between cancer and host cells may accelerate the growth of PDCD4-PRMT5 tumors. The increase in necrosis, edema, and vasculature is consistent with the larger size of the PDCD4-PRMT5 xenograft tumors, but further investigation is needed to determine whether any of these features are critical to the altered tumor growth properties conferred by elevated levels of PDCD4 and PRMT5.
The change in PDCD4 function that occurs in the presence of elevated PRMT5 pointed to a role for post-translational modification. We found that indeed PDCD4 is methylated and further, a PDCD4 methyl mutant expressed with wild type PRMT5 or a catalytically dead PRMT5 expressed with wild type PDCD4 failed to promote tumor growth. This demonstrates that methylation of PDCD4 by PRMT5 is critical for enhanced tumor growth. The methylation target residue, R110, lies near a S6 Kinase 1 site (S67) reported to regulate PDCD4 stability (26
). Levels of ectopic expression of PDCD4 did not appear to be influenced by the presence of PRMT5 or mutation of R110 ( and ). Furthermore, under conditions that stimulate PDCD4 degradation, the methyl mutant did not enhance or decrease stability indicating that methylation of PDCD4 does not help coordinate proteasome targeting.
PDCD4 is thought to exert its tumor suppressor role by regulating translation (21
), fitting with a growing theme of translation initiation as a node of regulation in cancer (31
). Interestingly, PRMT5 has been shown to influence eIF4E levels (32
) and was recently shown to influence ribosomal stability by methylation of ribosomal subunit RPS10 (33
). Methylation of PDCD4 may impact its role in translation regulation, although we did not observe a difference in eIF4A1 binding activity when the methylation site was mutated. Another interesting possibility is that, conversely, methylated PDCD4 changes PRMT5 function or specificity. In addition to the recent connection to translation, PRMT5 is known to be involved in transcription (13
), in efficient assembly of the spliceosome (35
), and in modulation of p53-dependent cell cycle arrest (32
). Methylated PDCD4 potentially accelerates tumor growth by altering these or other functions downstream of PRMT5 (32
By integrating biochemical and tumor model data, we have found that an elevated level of PRMT5 in conjunction with PDCD4 reverses the tumor suppressive properties of PDCD4. The finding that combined expression analysis of PDCD4
is a powerful prognostic indicator for outcome in breast cancer suggests that these factors could be used as rational, activity-based biomarkers to aide in decisions about how aggressively to treat a breast cancer patient. In the future, chemical inhibition of PRMT5 methyltransferase activity could be used to abrogate the synergism between PDCD4 and PRMT5, potentially unmasking PDCD4 tumor suppressor function in cancers that express both proteins. Finally, although our focus here has been on breast cancer, PDCD4 plays a tumor suppressor role in a wide spectrum of cancers (4
), raising the possibility that its connection to PRMT5 will be of broad prognostic, and perhaps therapeutic, value.