Recent studies have identified PI3K/Akt/mTOR pathway as a major oncogenic cascade for targeting molecular therapies in cancer25
. MTOR signaling has been implicated in the initiation and progression of multiple tumors, such as leiomyosarcomas and gliomas26
. We demonstrate herein that mTOR pathway is activated in a subset of patients with early HCC. Activation of mTOR cascade resulted from ligand-dependant signals from EGF and IGF signaling, rather than from a mutation-dependent mechanism, since no high-level amplifications and only marginal mutation rates in the most prevalent hot spots in PTEN, PI3KCA and PI3KB were identified. In fact, in a subset of patients, high levels of EGF could be primarily responsible for RPS6 activation. Coincidentally, down-regulation of the tumor suppressor PTEN was observed in a significant proportion of patients, mostly in advanced stages of the disease. All these results highlight the relevance of mTOR signaling in HCC, a pathway that has been insufficiently explored in human liver cancer.
These data are the first to characterize the status of RICTOR
status in human HCC. RICTOR
is part of MTORC2, however its functions and molecular structure are not entirely known27
. We found a significant association between gains in RICTOR
(25% of HCC) and its transcript expression. Intriguingly, gains in RICTOR
were significantly associated with p-mTOR, which could be a relevant mechanism of MTORC2 activation in human cancer. Genes significantly dysregulated in samples with gains in RICTOR (e.g. EGR2 and AMPK subunits) suggests that mTORC2 activity could influence signaling through the PTEN-mTORC1 axis. In vitro, downregulation of RICTOR by siRNA reduced cell viability of Huh7 cells, which harbor gains in RICTOR.
We speculate that in HCC, other mechanisms besides mTORC2 activation may be responsible for Akt phosphorylation at residue 473, since no correlation between p-Akt staining and gains in RICTOR
was observed. Membranous localization of p-mTOR was visibly lost in liver cancer; whether this event is due to divergent mTOR complex 1 and 2 cellular localization requires further investigation. Finally, a significant association between gains in RICTOR
and early recurrence was identified in the training set, a finding that requires further validation. Strikingly, and in accordance with this data, a recent report indicates that MTORC2 is hyperactivated in gliomas and functions in promoting tumor cell proliferation and invasive potential28
. Overall, these results suggest a possible role of MTORC2 in human hepatocarcinogenesis and warrant further evaluation of RICTOR
as a potential new target in HCC therapy.
Apart from the potential role of RICTOR
as an oncogene in liver carcinogenesis, we describe other mechanisms underlying mTOR pathway activation. We have confirmed dysregulation of key genes of the mTOR pathway (e.g. IGF2
) in HCC29, 30
, and herein reported dysregulation of other genes that includes mTOR and RAPTOR. Alterations in copy number or somatic mutations of PTEN, PI3KCA and PI3KB were not identified as major mechanisms of mTOR pathway dysregulation, although these genes were only assessed for mutations in certain exons. The infrequent mutation rate of PTEN (<10%) in earlier studies31
, supports our data, and suggests that other mechanisms apart from missense mutations may be responsible for PTEN downregulation (e.g. promother methylation32
). There is some controversy regarding PI3KCA mutations, with frequency rates that range from 0%33
in HCC. We did not identify any mutation in 2 exons of the PI3KCA gene.
The associations found at the protein level between p-EGFR/EGF and p-RPS6 suggest that RPS6 activation in HCC is partly driven through EGF signaling. Interestingly, our cohort showed higher rates of p-RPS6 activation than p-Akt, and similar to previous reports13
, there was not a significant correlation between both proteins, which reinforces the concept that alternative pathways besides Akt are responsible for the activation of MTORC1 (e.g. LKB1/AMPK signaling35
or direct signals from IGF-IR activation). Interestingly, marker genes significantly up-regulated in RPS6 activated HCC were related to NF-Kappaβ signaling (ZDHHC13
), angiogenesis (VEGFB
) and MAPK signaling (MAPK13
), highlighting the complex signaling cross-talk that is developing even at early clinical stages.
The primacy of mTOR activation in early HCC persisted in both cohorts analyzed, but was not associated with specific etiological factors. We found similar rates of p-RPS6 staining as previously described, but p-RPS6 in our cohort was a predictor of poor prognosis13
. Recent studies conducted by our group have identified, by unsupervised clustering of microarray data, a singular subgroup of HCC patients (proliferation group
) where activation of IGF and mTOR (p-RPS6) were enriched17
. This group has been previously associated with poorest outcome36
. In our series, patients with mTOR activation showed higher levels of AFP, less differentiated tumors, and a higher incidence of recurrence. These data highlight the relevance of this pathway as an attractive target for the development of new anti-tumoral agents.
To address the issue of multi-targeted blockade of mTOR signaling in HCC and neutralization of potential oncogenic loops, we evaluated the effect of a dual-level blockade of mTOR by an mTOR inhibitor (everolimus, RAD001) and an EGFR/VEGR inhibitor (AEE788) in experimental models. The EGFR inhibitor induced high rates of apoptosis in vitro, a phenomenon not observed with the rapamycin analog. Although everolimus has already proved antitumoral effects in a rat model of HCC14
, our in vivo studies in mouse xenografts revealed an additive anti-tumoral effect with survival implications after dual-level blockade of mTOR pathway. Regarding anti-target activity, RAD001 and AEE788 effectively blocked EGFR and RPS6 phosphorylation, respectively, in vitro
and in vivo
. Unlike everolimus, the EGFR inhibitor significantly increased apoptosis based on FACS analysis, PARP cleavage and TUNEL staining. Everolimus exerted its antineoplasic activity by inducing G1 arrest and hence inhibiting proliferation. Therefore, this combination of pro-apoptotic and anti-proliferative mechanisms may explain the additive effect obtained in the experimental model. Recent reports demonstrate that rapalogs inhibit mTORC2 activity in vitro37
, so we cannot exclude additional antitumoral activity related to this mechanism.
The recent breakthrough in the management of HCC by using the muti-kinase inhibitor sorafenib has established the concept of extending survival by using molecular targeted therapies in this otherwise chemo-resistant disease. Sorafenib provides three months survival extension in patients with a base-line median survival of 8 months5
. These data have stimulated the search for additional combination therapies, recognizing that HCC is an heterogeneous cancer with many genetic aberrations. In the present study we establish that mTOR pathway activation plays a role in HCC and that RICTOR may be a mediator of human hepatocarcinogenesis, although further evidence will be required to characterized RICTOR as a new oncogene in human cancer. Up-stream ligand-dependant mechanisms might also be responsible for mTOR pathway activation, indicating that this cascade offers relevant targets for cancer drug discovery. The combination of rapamycin analogs and EGFR inhibitors provides evidence for a proof-of-concept effect in experimental models of HCC. Consequently, these data provides the rationale to test combination therapies in early clinical trials in human liver cancer that includes RAD00138
. These studies should follow the guidelines reported on design and endpoints in clinical trials in HCC22