Individual patient-derived primary cells showed a wide range of sensitivity to metformin fig. S2, A and B. Titration of syrosingopine in the presence of 5 mM metformin in 12 samples showed synergistic cell killing in all cases Fig. Blast cells from 3 patients were metformin-sensitive compared to the previous 12 patients fig. S2B , but in one case AML , it was still possible to perform a cotitration with syrosingopine at a lower metformin concentration 2. This also turned out to be responsive to the drug combination fig. Thus, all primary leukemic cells tested 13 of 13 responded to syrosingopine-metformin treatment.
Peripheral blood cells from healthy blood donors were insensitive to metformin and to syrosingopine-metformin treatment Fig. Two nontransformed cell lines generated from primary human skin fibroblasts, Fib3 and Fib4, were also nonresponsive to the drug combination, suggesting that the effect is specific to transformed cells fig. The mode of cell death occurred via induction of apoptosis, as measured by annexin V staining, an early apoptotic marker, with first indications of cell death observed by 20 hours after treatment Fig.
This was confirmed with an alternative assay, measuring cell viability, cytoxicity, and caspase activity fig. Annexin V staining of blast cells from one leukemic patient CML confirmed that, in primary human leukemic blasts, the mode of killing was also due to induction of apoptosis Fig. Hepatospheres are three-dimensional 3D cultures of hepatocytes that more accurately mimic the in vivo conditions of solid tumors compared to conventional monolayer cell culture We generated hepatospheres from the hepatocellular carcinoma lines Huh7 and HepG2, which are responsive to the syrosingopine-metformin combination in monolayer cell culture table S1.
The drug combination, when added 24 hours after sphere formation, caused disaggregation of fully formed hepatospheres and killing of individual cells Fig. However, the amount of syrosingopine and the length of treatment were greater than those required for killing of the cell lines in 2D cultures. A Hepatospheres of Huh7 hepatocellular carcinoma cells treated with syrosingopine and metformin as indicated.
Quantitation of surviving cells by resazurin staining. C External liver appearance after 2 weeks of drug treatment. D Histological sections from vehicle- and drug combination—treated livers and accompanying pathological report. The in vivo efficacy of the drug combination was tested in a mouse liver cancer model.
The liver is greatly enlarged, and tumor nodules develop at multiple foci, starting from 12 weeks of age. Already after this short treatment, there was a reduction in liver size and the number of visible tumor nodules Fig. Histological examination of liver sections showed a reduction in tumor burden.
Syrosingopine sensitizes cancer cells to killing by metformin
The pathological report classified combination-treated mice as having fewer and smaller nodules with large necrotic areas that were absent in vehicle-treated controls Fig. Livers from two combination-treated mice were classified as tumor-free. Complex I inhibition by metformin has been shown to have a direct antitumor effect Phenformin, a more potent analog of metformin, has been proposed as an alternative to metformin in the frame of anticancer therapy Phenformin also synergized strongly with syrosingopine fig. S5A , suggesting a common mode of action with metformin in eliciting synthetic lethality with syrosingopine, namely, via complex I inhibition.
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Next, we tested syrosingopine in combination with an array of pharmacological agents that inhibit the ETC at different points, comprising inhibitors against all four ETC complexes, an inhibitor of the F 1 F O ATP synthase, and a proton ionophore [carbonyl cyanide p -trifluoromethoxyphenylhydrazone FCCP ] that dissipates the mitochondrial membrane potential. Cell death was induced in all cases, indicating that synthetic lethality is indeed due to inhibition of mitochondrial ETC activity and not via an extramitochondrial off-target effect of the biguanide drugs metformin and phenformin Fig.
A Proliferation assay of 6. Data shown are non-normalized, and the y axis intercept shows the effect on cell growth of each mitochondrial inhibitor by itself in the absence of syrosingopine. B Immunoblot of 6. C Proliferation assay of 6. Growth was measured after 3 days of treatment. To independently corroborate these findings, we generated cells lacking the mitochondrial genome. These results allow us to generalize that syrosingopine is synthetic lethal with inhibition of mitochondrial electron transport.
Metformin and its more potent analog phenformin have attracted interest as potential anticancer agents However, the concentrations required for anticancer efficacy in preclinical models are greater than the plasma concentration attained with typical dosing in diabetic patients Similar titrations for 6. HL60 cells were treated for 20 hours with the following compounds: C Mitochondrial membrane potential of cells treated with the indicated drugs for the indicated length of time measured by TMRM staining. Viability of syrosingopine-metformin—treated cells was measured by trypan blue staining, followed by automated cell counting.
The low concentrations at which the abovementioned mitochondrial inhibitors elicit synthetic lethality would be expected to have only a slight impact on mitochondrial function. To confirm this, HL60 cells were treated with the mitochondrial inhibitors at the concentrations used for synergistic killing with syrosingopine, and the mitochondrial membrane potential was measured using the potentiometric dye TMRM tetramethylrhodamine methyl ester. After 20 hours of treatment, there was no change in membrane potential compared to the untreated control Fig.
Syrosingopine alone had no effect on the membrane potential in HL60 cells except in combination with metformin, when it resulted in a collapse in the membrane potential after 20 hours of treatment Fig. Syrosingopine-metformin treatment for a shorter period 6 hours in HL60 did not generate a drop in the membrane potential.
In nonresponsive HT and NA8 cells, treatment with the syrosingopine-metformin combination had no effect on the membrane potential. Depolarization is correlated with low cell viability, as measured by trypan blue staining Fig. Although this may suggest that depolarization is involved in eliciting cell death, it could also be the case that the depolarized cells detected are those already undergoing cell death as a consequence of the combined drug treatment.
It remains possible that syrosingopine is acting by preventing the maintenance of mitochondrial membrane potential. In the presence of respiratory chain inhibition, the function of the F 1 F o -ATP synthase is reversed, and it uses cytosolic ATP generated by glycolysis to pump protons across the mitochondrial inner membrane to maintain the membrane potential 31 , Thus, any inhibitory effect by syrosingopine on the reverse F 1 F o -ATP synthase activity would be sufficient to elicit synthetic lethality in the presence of respiratory chain inhibition.
This membrane potential is oligomycin-insensitive but azide-sensitive, as has been reported by Appleby et al. However, the membrane is not depolarized by syrosingopine, leading us to discount syrosingopine-dependent inhibition of F 1 F O -adenosine triphosphatase activity as a potential mechanism for its mode of action. To summarize, neither syrosingopine nor the mitochondrial inhibitors, when applied singly at the low concentrations used to elicit synthetic lethality, have a detectable effect on mitochondrial function as measured by the membrane potential.
Nevertheless, this weak ETC inhibition is potentiated by syrosingopine to generate strong synergistic killing, giving hope that this may be clinically effective in combination with low plasma biguanide concentrations. Syrosingopine is derived from the potent antihypertensive drug reserpine fig. Both drugs inhibit the vesicular monoamine transporters VMAT1 and VMAT2 to prevent sequestration of monoamines into their storage granule, thus depleting catecholamine stores 33 , Syrosingopine is less potent than reserpine, with consequently weaker antihypertensive activity 35 , To test whether syrosingopine elicits synthetic lethality via VMAT inhibition, we tested the combination of reserpine with metformin.
Despite being a stronger VMAT inhibitor, reserpine only weakly synergized with metformin and was less potent than syrosingopine in inducing synthetic lethality Fig. To confirm that synthetic lethality with metformin is not due to VMAT inhibition, we performed a cotitration of metformin with tetrabenazine, a potent VMAT inhibitor structurally unrelated to the reserpine-like compounds fig. Tetrabenazine had no synthetic lethality with metformin in 6. Collectively, these data suggest that synthetic lethality with metformin by syrosingopine is unrelated to VMAT inhibition.
We used DARTS drug affinity responsive target stability to identify the binding partners of syrosingopine We observed a prominent band that was protected from proteolytic digestion in the presence of syrosingopine Fig. Band marked with asterisk was excised, and proteins were eluted for mass spectrometry. D Enolase activity assay performed at room temperature for HL60 lysates treated as indicated. E Measurement in 6.
RLU, relative luminescence units. Enolase activity was measured in HL60 lysates by an in vitro activity assay. However, we could not detect any inhibition of enolase activity by syrosingopine, although it was inhibited by the known enolase inhibitor NaF Fig.
The effect of syrosingopine on glycolysis at the cellular level was monitored using 6. Most cells are metabolically flexible and can switch between glycolysis and mitochondrial oxidative phosphorylation for ATP production, should either one of the pathways be inhibited. As expected, metformin had no effect on glycolytic output in this background Fig.
Thus, despite the lack of enolase inhibition seen in the in vitro assay Fig. To see whether enolase inhibition elicits synthetic lethality with metformin, a NaF-metformin titration was performed Fig. NaF was toxic between 2 and 10 mM, which was not increased by the addition of metformin. The absence of synergy between NaF and metformin suggests that the strong synthetic lethality of syrosingopine in combination with metformin is due to more than just the simultaneous inhibition of glycolytic and mitochondrial ATP-generating pathways.
There are three enolase isozymes in humans: E Proliferation assay of Colo cells titrated with syrosingopine in the presence or absence of 4 mM metformin for 4 days. Eno2 overexpression in the responsive cell line OPM2 Fig. Selecting for resistant cells enriched for Eno2-expression that are resistant to syrosingopine-metformin treatment Fig. The resistant cells had a lower proliferation rate, and removal of selection pressure resulted in loss of high Eno2 expression in the cell pool Fig.
Thus, whereas ectopic Eno2 expression in a previously sensitive background confers resistance to the drug combination, the reverse does not hold, and loss of Eno2 does not render cells insensitive to syrosingopine-metformin treatment. We describe a potent interaction between syrosingopine, an antihypertensive drug, and mitochondrial ETC inhibitors.
These two classes of drugs, when combined, elicit a synthetic lethal reaction in most of the cancer cell lines tested, which was specific to transformed cells. A Protocol used to examine the effect of rictor deletion on tumor development and maintenance. Top, for carcinogenesis experiments, rictor deletion was induced prior to nmol DMBA initiation with topical application of 4OHT daily for five treatments.
B—D Effect of rictor deletion prior to initiation on tumorigenesis. B Percentage of mice with papillomas. Previous results have demonstrated that treatment of tumor-bearing mice with rapamycin rapidly decreased tumor burden 24 , The results demonstrate that 4OHT-induced rictor disruption in tumors inhibited their further growth Figure 4. These results clearly indicate that inducible disruption of rictor in established tumors leads to their regression.
Effect of rictor deletion in established tumors. Dimensions of tumors were measured prior to 4OHT treatment and weekly thereafter. Mice continued to receive TPA twice weekly. A Waterfall graph showing the percent change in surface area for each individual tumor relative to its initial size. Left , typical keratinocyte-derived exophytic lesions of the skin in both experimental groups. Right, tumors were analyzed for expression of CC3 as described in the Materials and Methods.
Results are representative of multiple tumors taken from 3—4 mice in each group. Total positive cells in four non-overlapping fields were counted for each tumor. BrdU incorporation was not different between the groups. To determine the mechanism of tumor regression in response to rictor deletion, mice were killed 4 weeks after the beginning of the regression experiment week 25 of the carcinogenesis experiment , and serial sections from paraffin-embedded tumors were analyzed. To establish whether loss of Rictor decreases tumor cell proliferation, mice were injected with BrdU and tumors were analyzed for S-phase cells.
This is consistent with the unaltered hyperproliferation response observed in the skin of Rictor-deficient mice after TPA treatment Figure 2.
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Taken together, these results demonstrate that loss of Rictor sensitizes cells exposed to UVB in vitro to caspase-dependent apoptosis. We further elucidated the role of mTORC2 in genotoxic stress by exposing iRicKO cells with and without rictor deletion to cisplatin, then performing clonogenic assays. Effect of rapamycin or Torin-2 treatment of wild-type and Rictor-deficient cells on UVB-induced cell death. Cells were harvested 24h later and cell viability was measured by MTS assay. Data are representative of 2 independent experiments.
The exception was at the h time point, when significant cell toxicity was observed data not shown. For each protein analyzed, samples from both treatment groups are loaded onto the same gel to allow direct comparison of protein expression levels. Phosphorylated proteins were normalized to their corresponding total protein on the same gel and other proteins were normalized to either Lamin B1 or Tubulin on the same gel, as described in the legend for Supplementary Figure 2 , available at Carcinogenesis Online.
One Lamin B1 blot and one Tubulin blot are shown for reference. Results are representative of 2—3 independent experiments.
Introduction
Silencing of Rictor expression in tumor-bearing animals triggered regression of established tumors accompanied by increased levels of apoptosis without changes in proliferation. In vitro studies suggest the increased sensitivity to apoptosis in the absence of Rictor is mediated through mTORC2-dependent control of p -Akt Ser signaling without alterations in pathways controlled by mTORC1.
These data provide strong evidence that mTORC2 is necessary for both skin tumor development and maintenance of established tumors. Previous work has established that Rictor plays an essential role in the developing embryo 3 , whereas conditional deletion of rictor in adipose tissue, liver and brain revealed the functional importance of mTORC2 in these organs 27 , 36 , To investigate the contribution of mTORC2 to normal epidermal homeostasis and skin carcinogenesis, we used a model in which rictor recombination is driven by the Keratin 14 promoter in a 4OHT inducible manner, resulting in loss of rictor in the basal layer of the epidermis.
We first showed that rictor deletion in adult epidermis caused no apparent defects in skin architecture and did not alter the proliferation response to multiple applications of TPA, despite a dramatic reduction in epidermal p -Akt S This is consistent with previous studies in Drosophila , where loss of Akt hydrophobic motif phosphorylation had little effect on normal tissue growth Similarly, Rictor-dependent phosphorylation of Akt S was shown to be non-essential in mouse skeletal muscle and normal prostate epithelium 26 , Because the absence of p -Akt S affects only a subset of Akt targets 40 , our results suggest that normal epidermal proliferation is dependent on substrates of p -Akt T , and consequently requires mTORC1.
Although signaling intermediates other than Akt are also involved in epidermal proliferation, our findings are in agreement with previous results showing that rapamycin potently inhibits TPA-induced epidermal hyperproliferation 24 , and that overexpression of a constitutively activated form of Akt in the skin causes a hyperplastic epidermal phenotype Using a chemical carcinogenesis protocol, we went on to investigate the effect of rictor ablation on skin tumor development. Thus, mTORC2 is necessary for skin tumor initiation, but not for keratinocyte proliferation.
Taken together, these data support the hypothesis that mTORC2 is essential to maintain high Akt signaling in these tumor types. However, this does not rule out the possibility that these tumors arose from initiated cells that escaped recombination. An equally important and novel finding in our study is that disruption of rictor after tumors had formed not only prevented further growth, but also led to tumor regression. Furthermore, this regression occurs without changes in proliferation but with increased cleavage of caspase-3, consistent with increased apoptosis.
These results suggest that mTORC2 plays a critical role in mediating keratinocyte survival pathways, and identify mTORC2 as a target for both prevention and treatment of non-melanoma skin cancer. Furthermore, since cutaneous squamous cell carcinoma often becomes resistant to standard cisplatin therapy 41 , our clonogenic data support the study of TORC2 inhibitors as a therapeutic option in the setting of cisplatin resistance.
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As an extension of our in vivo studies, we demonstrated that cells with loss of rictor are sensitized to caspase-dependent apoptosis in response to carcinogen exposure in vitro. It is known that mTORC2 phosphorylation of Akt at Ser promotes cell survival through a number of downstream effectors 2—6 , Rictor has been reported to act independently of mTORC2 through functional interactions with a number of proteins.
The Rictor-Cullin 1 complex acts as an E3 ubiquitin ligase to control degradation of the serum and glucocorticoid-inducible kinase It has also been found that a complex between Rictor and integrin-linked kinase can regulate Akt S phosphorylation and cell survival in the absence of mTOR in a subset of human breast cancer cell lines In summary, these studies show for the first time that, while dispensable for normal keratinocyte proliferation, mTORC2 is essential for both skin tumor development and maintenance of established tumors.
They further suggest that mTORC2 controls pro-survival pathways both in vitro and in tumors. Although the development of mTORC2-specific inhibitors has been elusive, our results support the idea that activated Akt signaling will render specific tumor types more responsive to therapy targeting the mTOR pathway.
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