Sorafenib is a small molecule drug that inhibits RAF family
serine / threonine kinases and class III receptor tyrosine kinases such as the PDGF and FLT receptors. Previous studies have shown that sorafenib and the histone deacetylase inhibitor vorinostat interact in vitro and in vivo in a greater than additive fashion to kill transformed cells that was mechanistically dependent on activation of CD95 (29
). The present studies attempted to determine in much greater detail the molecular mechanisms by which sorafenib and vorinostat, as individual agents, interacted to activate CD95 and promote drug toxicity.
Sorafenib (3-6 μM) caused a dose-dependent increase in CD95 tyrosine phosphorylation and, based on multiple criteria, in parallel activated Src family non-receptor tyrosine kinases. Expression of dominant negative Src or knock out of Src / Fyn / Yes blocked sorafenib –induced CD95 tyrosine phosphorylation. CD95 (Y232F, Y291F) was not activated by either higher sorafenib (6 μM) concentrations or by sorafenib (3 μM) and vorinostat treatment. Knock down of class III receptor tyrosine kinase PDGFRβ expression enhanced Src activity; in a Src-dependent fashion knock down of PDGFRβ enhanced CD95 tyrosine phosphorylation and also suppressed the ability of sorafenib to induce CD95 tyrosine phosphorylation. Vorinostat rapidly enhanced the ability of lower sorafenib concentrations to increase ROS levels and to transiently suppress total cellular PTPase activity within 1h that was due to ROS generation. However, ROS generation also significantly enhanced PTPase activity 3h after drug treatment and correlated with Src Y527 dephosphorylation and with vorinostat-stimulated CD95 tyrosine phosphorylation. Thus molecular one potential model for drug action is that sorafenib-mediated inhibition PDGFRβ causes a compensatory activation of Src family tyrosine kinases that in turn phosphorylate and activate CD95. And, based on the degree to which PDGFRβ is inhibited by sorafenib, CD95 becomes tyrosine phosphorylated in a Src dependent fashion. Vorinostat, by interacting with sorafenib to elevate ROS levels rapidly suppresses cellular PTPase activity that promotes greater levels of CD95 tyrosine phosphorylation and CD95 activation.
Knock down of PDGFRβ increased basal plasma membrane levels of CD95 that was Src dependent and when PDGFRβ was knocked down, the relative ability of sorafenib to cause further activation of CD95 was reduced. This finding was mirrored by changes in autophagy presented in wherein knock down of PDGFRβ increased autophagy in a CD95 dependent fashion and in cells lacking PDGFRβ the ability of sorafenib to cause additional autophagy was reduced. And, although we observed enhanced levels of CD95 activation after PDGFRβ knock down in , this did not translate into increased basal levels of cell killing in ; possibly this was because we were observing elevated levels of protective autophagy after knock down as was shown in (29
). Thus, logically, based on data in , the relative reduction in sorafenib-induced cell killing in PDGFRβ knock down cells in is because without expression of the key target, i.e. PDGFRβ, sorafenib cannot instantaneously initiate the series of events that will lead to PDGFRβ inhibition, compensatory Src activation, CD95 tyrosine phosphorylation etc.
Based on our data, if sorafenib is promoting activation of Src family kinases, we reasoned it is probable that other targets of Src kinases are also phosphorylated in response to sorafenib treatment. For example, in a preliminary study we have noted that FAK and IGF1R tyrosine phosphorylation are enhanced in response to sorafenib exposure. Increased FAK signaling has the potential to promote metastatic spread of tumor cells via Src (39
). Signaling by Src kinases is known to facilitate ERBB1 Y845 phosphorylation and we observed this in our system following sorafenib treatment. Phosphorylation of Y845 has been linked to ERBB1-induced tumor cell growth, independent of ERK1/2 and PI3K signaling (40
). In at least one study, low concentrations of sorafenib have been shown to promote MEK1/2 activation via IGF1 receptor signaling, and inhibition of MEK1/2 signaling enhanced sorafenib toxicity (41
). We have recently published similar data in malignant blood cancer cells (42
). Thus the practical outcome of sorafenib promoting Src kinase activation is that whilst this drug acts to suppress tumor growth through Src-dependent activation of CD95, the induction of endoplasmic reticulum stress and inhibition of pro-angiogenic growth factor receptors; it also has the capacity to promote tumor cell survival and migration through elevated signaling by Src, FAK, ERBB1, IGF1R and possibly the ERK1/2 pathway. Further studies will be required to define the precise nodal enzymes within the sorafenib-induced signaling network that can promote and suppress sorafenib lethality.
We recently noted using higher concentrations of vorinostat than those used in this manuscript that the drug induced DNA damage, via ROS generation, and this played a key role in the activation of NFκB by the drug (43
). Vorinostat facilitated CD95 activation in hepatoma cells by two mechanisms: increased CD95 tyrosine phosphorylation and increased expression of FAS-L. Vorinostat –induced ROS generation, and when in combination with sorafenib, inhibited PTPase activity and then profound activated PTPase activity; effects that were both ROS dependent. These effects also correlated that correlated with increased CD95 phosphorylation, increased Src Y416 phosphorylation and dephosphorylation of Src Y527. PTPases have a ~10-fold higher specific activity than the protein kinases whose actions they reverse meaning that a small reduction in PTPase activity can significantly modify a site of phosphorylation whose phosphorylation is being actively increased due to actions of a kinase (44
). The precise PTPase that regulates CD95 tyrosine phosphorylation or the Src Y527 phosphorylation in hepatoma cells has not been identified although it has been shown that both CD45 and SHP1 can proximally modulate CD95 signaling in immune cell types, and that both SHP1 and Src family kinases can associate with CD95 (45
). This data, combined with our present findings, argues that specific inhibitors of SHP-1 could be of value in promoting sorafenib toxicity.
Using a neutralizing antibody, inhibition of FAS-L function suppressed sorafenib + vorinostat toxicity by ~50%. This correlated with vorinostat, in an NFκB –dependent fashion, promoting increased expression of FAS-L 12-24h after drug exposure. However, a multitude of studies have shown that NFκB activation can also stimulate expression of anti-apoptotic proteins and promote cell growth. Previously we have shown that the rapid (~6h) –induced activation of CD95 was due, in part, to ceramide generation and our present findings demonstrated that CD95 activation at this time point was insensitive to the neutralizing antibody. Multiple HDACIs have demonstrated to increase protein levels of FAS-L and/or CD95 (e.g. refs. 38
), and this has been suggested as one important mechanism for the anti-tumor effects of HDACIs.
One well-described additional action of HDACIs in tumor cells is to increase expression of the cyclin dependent kinase inhibitor p21Cip-1/WAF1/mda-6
). We noted that vorinostat, in contrast to our findings in malignant blood cancer cells, modestly increased p21 levels in hepatoma cells and that this effect was suppressed in cells treated with vorinostat and
sorafenib. Increased expression of p21 not only acts to suppress tumor growth but in some cell types suppresses toxic JNK pathway signaling and also facilitates CD95 activation (36
). Knock down of p21 expression suppressed vorinostat and sorafenib toxicity and suppressed drug combination-induced autophagy that was associated with reduced CD95 activation. We also noted that loss of Src function blocked sorafenib (6 μM) –induced protective autophagy; autophagy that was CD95 dependent. In contrast, in malignant blood cancer cells we have previously shown that expression of p21 suppressed the lethality of vorinostat and sorafenib treatment (20
). Thus vorinostat as a single agent has the potential to kill tumor cells by increasing FAS-L and p21 levels but also could act to promote tumor cell survival through NFκB activation. Thus sorafenib, in part, subverts vorinostat –induced cell killing processes by blocking increased p21 expression. Of note was our finding that sorafenib suppressed vorinostat –induced p21 expression but not the enhancement of FAS-L expression; as p21 protein levels, unlike those of FAS-L, are regulated by both protein and mRNA stabilization as well as for both proteins at the level of transcription, our data suggests that the HDACI –induced expression of p21 most probably occurs at a post-transcriptional level (48
We have previously demonstrated that sorafenib and vorinostat combination therapy are effective at killing hepatoma cells in vivo, and sorafenib and HDACI combination therapy is entering phase I evaluation in hepatoma, and also in renal carcinoma and non-small cell lung cancer, patients (29
). The present studies provide additional mechanistic information as to how these agents interact and, furthermore, predict that inhibitors of survival signaling receptors e.g. ERBB1 and IGF1R, which are activated in response to sorafenib exposure, will enhance the anti-tumor efficacy of this drug combination.