MTOR signaling is frequently activated through several different mechanisms by upstream tyrosine kinase membrane receptors (RTK) which promote tumor growth and survival signaling. Protein expression of several of these RTKs was examined by western blot () in four RCC cell lines, two of which are VHL mutant (Caki-2 and UMRC3). Platelet derived growth factor beta (PDGFRβ) was expressed at high levels in all 4 cell lines examined, epidermal growth factor receptor (EGFR) was predominantly expressed in A498 and UMRC3, vascular endothelial growth factor receptor (VEGFR) demonstrated higher expression in A498 and Caki-2, and insulin growth factor receptor (IGFR) was found to be highly expressed in Caki-1, Caki-2, and UMRC3. Phosphorylation of mTOR at serine 2481 indicates activation of the mTORc1 complex. A498, Caki-1 and Caki-2 demonstrate high levels of phosphorylated mTOR, and UMRC3 to a lesser degree (). Dose response of temsirolimus (0.1–150 nM) demonstrated little growth inhibitory activity in all four RCC cell lines (). Temsirolimus treatment of cells for 24 hours at a 10 nM dose strongly down-regulated phosphorylated mTOR (p-mTOR) in all cell lines, except for Caki-2, which showed some resistance to temsirolimus (), indicating the drug is successfully abrogating mTORc1 activity. Thus, temsirolimus effectively blocked active p-mTOR, but had little growth effect upon four RCC cell lines when used alone.
Figure 1 Temsirolimus down-regulates p-mTOR in RCC cell lines. A. Western blot panel of 4 RCC cell lines examining receptor and mTOR expression. B. Cell proliferation assay showing that temsirolimus after 72 hours does not have a dose effect. C. Western blot analysis (more ...)
In order to produce an anti-tumor growth response in RCC cells, the chemotoxic microtubule stabilizing drug Ixabepilone was examined for efficacy. Dose response curves demonstrated an IC50 of ~ 2 to 3 nM for ixabepilone for all four cell lines (). In order to verify that ixabepilone was effectively stabilizing the microtubules, a microtubule stabilization assay was performed as shown by western blot. β-III tubulin and its binding partner, α-tubulin, accumulated in the polymerized fractions (P) after 6 hour exposures to ixabepilone ().
Figure 2 Ixabepilone dose responsively inhibits cell proliferation in RCC cell lines. A. Cell proliferation assay showing that ixabepilone has a dose effect after 72 hours and IC50 was determined to be ~ 2 nM overall. B. Western blot analysis of cells treated (more ...)
Since RCC cell lines were resistant to temsirolimus despite downregulation of p-MTOR, but sensitive to ixabepilone, the two chemotherapeutics were used in combination to examine synergistic effects (). A dose range of ixabepilone (0.1-3 nM) was combined with fixed doses of Temsirolimus (0, 0.1, 1, or 10 nM). Overall, temsirolimus and ixabepilone are synergistic when combined at low concentrations (nM) as shown by Fa-CI plots (). Dose ratios that did not exhibit synergy (>1) are marked by black circles in the plot legend. Interestingly, the renal cell line (Caki-1) that had complete loss of p-MTOR in response to temsirolimus (), exhibited the highest synergism as indicated by the Fa-CI plot (). The cell line (Caki-2) that was most resistant to p-mTOR downregulation (), only exhibited synergism when ixabepilone was used at concentrations higher than 1 nM ().
Figure 3 Combinatorial effects of temsirolimus and ixabepilone in RCC. Synergy curves using various concentrations of ixabepilone were used alone and in combination with three fixed concentrations of temsirolimus (0.1 nM, 1 nM, and 10 nM). Drug interactions were (more ...)
In order to identify whether the synergistic decrease in proliferation observed was due to decreased cell doubling or induction of cell death, cell death via propidium iodide staining as measured by flow cytometry was examined using 10 nM temosirolimus and 2 nM ixabepilone (). Again, Caki-1 cells showed the highest combinatorial effects at 38.4% cell death (). Caki-2 cells, which previously showed the most resistance to Temsirolimus (, ), had 29.3% cell death in combination (). UMRC3 cells were more resistant to cell death with only a 1.6% difference in the combination when compared to ixabepilone alone, suggesting that synergy may be due to lower proliferative capacity and not induction of cell death (). Western analysis confirmed that cell death due to Ixabepilone and combination treatment in all four RCC cell lines was due to apoptosis as indicated by PARP cleavage (). Combination treatment demonstrated elevated PARP cleavage when compared to all other treatment groups, with UMRC3 having the lowest levels of cleavage, reflective of the propidium idodide staining (). Of note, Caki-1 and Caki-2 both demonstrated significant cell death via propidium iodide staining (, ) due to Temsirolimus monotherapy, however no corresponding PARP cleavage (). This suggests that temsirolimus monotherapy may be inducing non-apoptotic mechanisms of cell death. Downstream targets of m-TOR were also examined and p-p70S6K was strongly down regulated with no change in p4EBP1 levels ().
Figure 4 Cell death combinatorial effects of temsirolimus and ixabepilone in RCC. Cells were treated with 10 nM temsirolimus (Tem) and 2 nM ixabepilone (Ixa) for 48 hours followed by cell death analysis as described in Materials and Methods. A. A498, B. Caki-1, (more ...)
Figure 5 Effects of temsirolimus and ixabepilone on RCC protein expression. Western blot panel of 4 RCC cell lines treated with 10 nM temsirolimus (Tem) and 2 nM ixabepilone (Ixa) for 24 hours. PARP and proteins downstream of p-mTOR were examined. All cell lines (more ...)
In order to identify mechanisms of synergistic cell death in RCC cells, downstream components of the mTOR signaling pathway and Ixabepilone were examined. Microtubule stabilizers such as Ixabepilone function by binding to microtubule polymers and preventing them from dissociating into monomers, thereby disrupting the process of MT treadmilling [29
]. MT dynamics are crucial for many cells processes, and a possible immediate consequence may be perturbation of endoplasmic reticulum (ER) homeostasis [30
]. MTOR signaling also plays a role in ER functionality through mTORC1 by mediating protein translation, metabolism, autophagy, etc [33
]. Thus, we examined ER stress as a convergence point of both Temsirolimus and Ixabepilone activity.
Combination therapy demonstrated the highest induction of endoplasmic reticulum (ER) stress signaling in both the Caki-1 (VHL wt) and Caki-2 (VHL mut) RCC cell lines treated with combinatorial therapy (10 nM temsirolimus and 2 nM ixabepilone) as shown by increased protein expression of downstream ER stress protein chaperone BIP and apoptosis inducer CHOP () when compared to control or monotherapy. ATF6 is one of the primary ER stress sensor, and translocates to the nucleus to upregulate transcription of several ER stress response proteins when activated, including BiP and CHOP [35
]. ATF6 expression was attenuated in Caki-1 and Caki-2 cells using two separate lentiviral shRNA constructs (ATF6-332 and ATF6-690) as shown by QPCR (). Both ATF6 shRNA constructs demonstrated significant recovery of cell proliferation in the presence of ixabepilone (2 nM) and temsirolimus (10 nM) combination therapy in both Caki-1 and Caki-2 cell lines compared to nontarget (NT) control (). We have thus demonstrated that ER stress likely mediates partof the antitumor synergy of combined mTOR inhibitor and a microtubule stabilizer.
Figure 6 Combination of temsirolimus with ixabepilone induces ER stress. Endoplasmic Reticulum (ER) stress was observed in Caki-1 and Caki-2 RCC cells treated with temsirolimus (10 nM) and ixabepilone (2 nM) as shown by A. induced expression of the ER stress markers (more ...)