Renal complications constitute the main cause of death among TSC patients (31–33). A significant portion of such complications include AML hemorrhage. Although end-stage renal disease is reported at low rates in TSC patients (34, 35), the studied populations are usually young. Notably, up to 40% of the TSC patients develop chronic kidney disease (CKD), exhibiting an estimated glomerular filtration rate equivalent to 30-year-older subjects from the general population (36). While the pathogenesis of TSC-associated CKD remains not completely understood, several factors are known to contribute to renal function decline, including tumor bleeding, tumor encroaching to normal surrounding renal tissue, renal cystic involvement, focal and segmental glomerulosclerosis and tubulointerstitial disease. A molecular mechanism involving TSC1 or TSC2 loss of function in renal parenchyma has also been proposed to play a role in reducing glomerular filtration rate (2, 37). Invasive interventions to prevent tumor hemorrhage, moreover, including partial nephrectomy and selective arterial embolization, can also impact on early loss of renal function as a consequence of loss of functional renal tissue (32). It should be noted that these invasive procedures are not uncommon in clinical practice (32, 36), given the potential lethality associated with AML hemorrhage. A recent databank study reported that 24.2% of TSC patients had at least one invasive kidney intervention.(38) Because TSC patients usually present multiple AMLs, and the incidence increases with age, such interventions usually cannot treat all lesions. Invasive procedures, therefore, are often repeated along life, leading to increased risk of CKD (32).
In response to this scenario, clinical studies have shown efficacy of mTORi in reducing AML volume, supporting these drugs as first-line therapy of asymptomatic AMLs larger than 3 cm in TSC patients (37). Such studies, however, had the primary end-point focused on tumor size reduction, not addressing whether the verified AML shrinkage lowers the bleeding rate and preserves renal function. Interestingly, however, no event of AML bleeding was reported in the extension of the phase 3 study “Everolimus for renal angiomyolipoma in patients with tuberous sclerosis complex or sporadic lymphangioleiomyomatosis (EXIST-2)” (39), a trial that followed 112 patients treated with everolimus for a median period of 28.9 weeks. This finding suggests a bleeding protective role of mTORi.
Despite the recognition of this effect of mTORi on AML volume, the response to these drugs has been shown to significantly vary among such tumors (39). In addition, AMLs are neoplasms histologically complex, with components derived from at least three distinct cellular phenotypes. Based on these two observations, we raised the hypothesis that mTORis might have different effects on distinct tumor components and compartments, and investigated this possibility in AMLs with different constitutions from a series of TSC patients. Of note, the elucidation of this effect might not only clarify a complex biological problem but also bring novel, important information to the clinical scenario. Clinical benefits could potentially include the development of predictors for AML response to mTORi as well as risk modifications associated with disease complications.
As expected, AMLs responded to sirolimus with volume reduction, a response that significantly varied among the tumors. Interestingly, our CT analyses revealed differential effects of sirolimus on AMLs with distinct proportions of fat. Fat-poor AMLs displayed a dramatic volume reduction response to sirolimus while such an effect was much milder in fat-rich tumors. In consistency with these findings, intermediate-fat AMLs presented an intermediate volume decrease behavior between fat-poor and fat-rich tumors. An important clinical derivative of this analysis was the finding that in AMLs fat poorness predicts a more effective response to sirolimus.
Our findings also revealed that the differential effect of sirolimus on AMLs is essentially based on differential effects on specific tumor compartments. Our data showed a primary reduction of the fat-poor portions of the tumors. Interestingly, the profound effect of sirolimus on the FPCs and HVCs promoted the transformation of some FPTs into FRTs, a finding not yet reported. The liposubstitution observed in such AMLs represents, in fact, the most striking translation of the remarkable differential actions of mTORi in the different AML compartments. Our findings also unraveled a dramatic decrease of vascular aneurysmatic formations. In line with this concept, the volume of the AML fat-rich compartment did not decrease, but instead increased, in response to treatment. Our findings of massive reduction of the vascular tumor components in response to sirolimus, in turn, suggest that this treatment is likely protective against AML bleeding.
Our data are in line with previous observations that in vitro effects of sirolimus differ among distinct cell lineages (40) and TSC patients display high levels of the pro-angiogenic molecules VEGF-A and VEGF-D (vascular epithelial growth factors A and D) (41). mTOR inhibitors, therefore, could differentially act in the different components of AML, with particularly high efficiency on vascular formations. mTOR complex 1 (mTORC1), in fact, is known to drive HIF-1α (hypoxia-induced factor 1α) and VEGF-A signaling via multiple mechanisms involving 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1), S6K1 (p70 ribosomal protein S6 kinase 1) and STAT3 (signal transducer and activator of transcription 3), an angiogenic process potentially attenuated by sirolimus (42). Interestingly, this mechanism is associated with acceleration of endothelial senescence (43). It is possible, therefore, that the molecular basis of the differential mTORi effects on the AML compartments is based on VEGF downregulation induced by sirolimus through inhibition of mTORC1 (44). Reduction of VEGF-D circulating levels was also reported in patients receiving rapamicyn.(24) The remarkable reduction of AML vascular components shown in our study suggests that mTOR activity is fundamental to the maintenance of such structures in AMLs. Moreover, since aneurysms are the main determinant for tumor hemorrhage(28), the inhibition of maintenance and development of aneurysmatic/ectatic formations induced by mTORi is the likely mechanism responsible for the reduction of bleeding occurrence observed in the EXIST-2 study and its extension (22, 39).