We demonstrated that while evidence of PI3K pathway activation is seen in both myxoid and round cell liposarcoma, such activation is significantly associated with round cell change. Tumors with round cell change were more likely to have strong membranous IGF1R expression or an activating mutation in PIK3CA
than purely myxoid tumors. Moreover, activation of the PI3K/Akt pathway in round cell tumors was verified by the increased expression of p4EBP1 compared to myxoid tumors. Increased p4EBP1 in both myxoid and round cell tumors was significantly associated with activating events upstream in the PI3K/Akt pathway (IGF1R expression, PTEN loss, or PIK3CA
mutation). Moreover, PIK3CA mutation and PTEN loss are mutually exclusive events, and were usually not seen in conjunction with increased IGF1R expression. Taken together, these findings support a role for increased flux through the PI3K/Akt pathway in round cell change, a form of tumor progression in myxoid liposarcoma. Our findings confirm and expand upon the findings of a previous high-throughput study demonstrating PIK3CA
mutations and activation of the PI3K/Akt pathway in myxoid and round cell liposarcoma.(17
In addition, we found differences in IGF1R, PTEN, p4EBP1, and pS6 expression between tumors with treatment response and tumors with myxoid or round cell morphology. One limitation of this finding is the paucity of viable tumor cells in treated tumors with extensive hyalinization. Interpretation may have been skewed by the presence of non-viable tumor cells or infiltrating cells such as histiocytes mimicking tumor cells. Nevertheless, the difference in p4EBP1 expression remained significant even when comparing only treated tumors with adipocytic maturation versus myxoid/round cell tumors. This suggests that the PI3K/Akt pathway is down-regulated in maturing tumor cells in response to therapy. However, residual foci of myxoid or round cell histology within treated tumors showed no significant difference in expression of pS6 or p4EBP1 compared to untreated tumors. The driving forces behind the response to therapy or lack thereof have yet to be elucidated.
Previously, Barretina et al.
described the clustering of PIK3CA
mutations in myxoid liposarcoma in exons 9 and 20, around codons 542, 545, and 1047, with mutations appearing in 13/71 (18%) of tumors.(17
). We found a similar percentage of mutations in our cohort (6/44, (13%)), although our analysis was limited to the 2 hotspots in exons 9 and 20 and could not identify the remainder of the reported rare mutations elsewhere in the gene. Our cohort possessed less frequent mutations in the helical domain (exon 9) and more in the kinase domain (exon 20) than were previously described. Of note, in the prior study of myxoid liposarcoma, H1047R mutations were reportedly not associated with increased pAkt; this was postulated to be due to high level PTEN expression in these tumors.(17
) We were unable to directly assess Akt phosphorylation status, as it was not robustly detectable in the formalin-fixed paraffin-embedded tissues used in this study. Nor did we find higher PTEN levels in tumors with H1047R mutation. This discrepancy may be due to the method of analysis (tissue immunohistochemistry vs. western blot). Nevertheless, the functionality of PIK3CA
mutations in our cohort is supported by elevated levels of p4EBP1 in these cases.
mutation was previously reported to be associated with shortened disease specific survival. While the authors did not report on myxoid vs. round cell histology,(17
) their finding would be in keeping with our discovery that PIK3CA
mutations are more frequent in the more aggressive round cell tumors. Increased pAkt has also been reported in round cell tumors,(16
) lending additional support to a role for the PI3K pathway in driving round cell transformation and tumor aggression.
We assessed two downstream targets of the PI3K pathway in order to confirm pathway activation. 4EBP1 is a translational repressor, known to be a direct target of the mTOR complex, and is inactivated by phosphorylation, (14
) while phosphorylation of S6 ribosomal protein is thought to regulate protein translation.(24
) Previous reports have indicated that S6 and 4EBP1 are equivalent markers of PI3K activation.(26
) However, we found that only p4EBP1 was associated with round cell liposarcoma or activating event in the PI3K/Akt pathway, while pS6 was only slightly elevated in these tumors. In fact, pS6 was most frequently seen at high levels in tumors with treatment effect, which were least likely to have evidence of an activating event in the PI3K pathway. One possible explanation for this finding is that is that S6 may be phosphorylated in tumors with treatment effect as part of the anti-apoptotic role of the PI3K pathway – a role that is known to involve the p70 S6 kinase responsible for the majority of S6 phosphorylation.(29
) Thus, elevated pS6 in treated tumors could represent cellular response to stress with activation of survival factors rather than an indicator of proliferation and transformation. Consistent with this theory, high levels of pS6 and p4EBP2 were each seen in 36% of tumors in our cohort without evidence of treatment effect and no known activating event in the PI3K pathway. Increased levels of both pS6 and p4EBP1 were then seen in tumors with activating events, although the change in expression only reached significance with p4EBP1. These findings suggest that in tumors without treatment effect, phosphorylation of S6 and 4EBP1 are both mediated by the PI3K pathway. However, the presence of phosphorylated S6 and 4EBP1 in 36% of tumors without IGF1R/PIK3CA
mutation/PTEN loss suggests that additional mechanisms of increased flux through the PI3K/Akt pathway are present in myxoid and round cell liposarcoma. Other activated receptor tyrosine kinases, including PDGFRB and EGFR, have been identified in myxoid liposarcoma,(16
) which may also lead to increased PI3K signaling. Additional studies are needed to further elucidate the myriad mechanisms by which PI3K/Akt signaling may be activated in these tumors.
No PIK3CA mutations were identified in 2 cell lines or 4 primary cell cultures from myxoid and round cell liposarcomas, although 1 of 4 primary cultures was found to have loss of PTEN expression. These cell lines and primary cultures could be used to model the role of the PI3K/Akt pathway in round cell transformation in myxoid liposarcoma, and to better understand the potential role of this pathway in tumor proliferation and invasion. Moreover, the primary cell culture with PTEN loss, and therefore activation of the PI3K pathway, may represent a valuable model in which to assess the efficacy of PI3K/Akt pathway inhibitors such as rapamycin or temsirolimus in treating these tumors. Additional study is needed to determine if the other cell lines and primary cultures may have other activating events in the PI3K pathway.
In summary, our findings posit a role for activation of the PI3K/Akt pathway in round cell transformation of myxoid liposarcoma. Mechanisms of activation include increased IGF1R, activating mutations in PIK3CA, as well as loss of PTEN. Preferentially increased levels of p4EBP1 are seen in round cell tumors, while pS6 may be by affected by additional factors. Additional studies are needed to further elucidate the role of PI3K pathway activation in driving round cell transformation. If our data are confirmed, they suggest that targeted therapy against factors involved in the PI3K/Akt pathway, such as mTOR, could someday play a role in treatment of aggressive round cell liposarcoma.