Mammary, hepatoma and lung carcinoma cells were grown in thymidine-less serum, conditions that replicate growth conditions of an in situ
tumor in a patient. Cells were treated with low clinically relevant doses of the anti-folate pemetrexed (PTX) and in response cells rapidly increased their levels of autophagy as judged by vesicularization of a green fluorescent protein tagged form of LC3 (ATG8) (LC3-GFP) and by increased processing of LC3 to LC3II ( and Figure S1
). Drug-induced autophagy was first observed at a higher pemetrexed dose within 6h of treatment and autophagy was noted at all doses within 12h. The induction of autophagy was blocked by a small molecule inhibitor of the class III PI3K Vps34, 3 methyl-adenine (3MA), or by knock down of Beclin1 or ATG5 (data not shown, Figure S2
). Within 24h, treatment of breast cancer cells with pemetrexed caused a dose-dependent reduction in cell viability as judged by Annexin-PI flow-cytometry, an effect that was blocked by 3MA or by knock down of Beclin1 ().
Pemetrexed induces autophagy and tumor cell killing that is suppressed by knock down of Beclin1 or treatment with 3-methyl adenine
Sorafenib (Sor) is a multi-kinase inhibitor whose biologic actions have often been tied to inhibition of class III receptor tyrosine kinases, e.g. vascular endothelial growth factor receptors (VEGFRs) and platelet derived growth factor receptor beta (PDGFRβ) (35
). We and others have also noted that sorafenib can stimulate autophagy through inhibition of class III RTK’s and that in a dose-dependent effect this response can either be a protective form of autophagy or a toxic form of autophagy (e.g. 35
). Hence, we next determined whether sorafenib enhanced, or suppressed, pemetrexed toxicity. Sorafenib and pemetrexed interacted in a greater than additive fashion to increase the number of autophagic vesicles in tumor cells that correlated with increased processing of LC3 to LC3II and that was inhibited by knock down of Beclin1 (). Sorafenib enhanced the toxicity of pemetrexed in a dose-dependent fashion in short-term 24h cell viability assays using multiple tumor cells from a diverse range of tissue types (; Figures S3–S7
). Knock down of Beclin1 blocked the pemetrexed + sorafenib drug combination -stimulated induction of autophagy and suppressed the cytotoxic interaction between sorafenib + pemetrexed (; Figure S8
). Incubation of cells with 3MA also suppressed pemetrexed + sorafenib –induced toxicity (data not shown).
Pemetrexed interacts with sorafenib in a dose-dependent fashion to increase autophagy and tumor cell killing that is suppressed by knock down of Beclin1
It has been noted by the Moran laboratory that pemetrexed treatment increases the intracellular concentration of the chemical ZMP (AICAr monophosphate; 5-Aminoimidazole-4-carboxamide-1-β-D-Ribofuranosyl monophosphate) resulting in ZMP-induced activation of the AMP-activated protein kinase (AMPK); AMPK activation in turn acts to suppress mTOR activity (1
). Reduced mTOR activity has been associated with elevated levels of autophagy. Because of these findings, we investigated whether suppression of mTOR function by use of rapamycin altered pemetrexed lethality (1
). Rapamycin (Rap) enhanced pemetrexed toxicity in multiple tumor cell types (Figure S9
). Thus suppression of the PI3K-mTOR pathway at the level of a growth factor receptor or at the level of mTOR could enhance pemetrexed lethality.
The development estrogen independence in recurrent mammary tumors; i.e. those tumors initially diagnosed as being ER+ and thus chronically treated with Tamoxifen, has frequently been observed. For ER+ postmenopausal breast cancer patients the development of other “purer” anti-estrogen therapeutics such as Faslodex (Fulvestrant, ICI 182,780) has also been beneficial though even these purer anti-estrogen modalities have potential to fail with the delayed outgrowth of estrogen independent breast cancer cells (42
). We next determined the impact of Fulvestrant resistance on the lethality of the pemetrexed + sorafenib drug combination in a well characterized ER+ breast cancer cell line; a cell line isolated from a pleural effusion, MCF7.
In agreement with a role for autophagy in the regulation of mammary tumor cell survival following sorafenib + pemetrexed, in parental MCF7 and fulvestrant resistant MCF7 cells (MCF7F) sorafenib or the drug combination increased LC3II processing and decreased p62 levels (). Parental MCF7 cells are known to be haplotype for expression of the autophagy regulatory gene Beclin1 and it has been postulated in clinical samples from breast cancer and lung cancer patients that loss of Beclin 1 facilitates mammary / lung tumorigenesis (e.g. 43
). Basal Beclin1 levels were significantly elevated in MCF7F compared to parental MCF7 cells; similar findings were also noted for ATG5-ATG12 levels. In parental MCF7 cells drug combination treatment increased Beclin1 and ATG5-ATG12 expression to approximately the same extent as the basal protein levels in MCF7F cells (). Beclin1 and ATG5-ATG12 levels were not further enhanced by the drug treatment in MCF7F cells.
Fulvestrant resistant MCF7 cells express higher levels of autophagy markers and mitochondrial protective proteins; protection of the mitochondria blocks pemetrexed + sorafenib toxicity
In addition to altering expression of autophagy regulatory proteins, drug combination exposure of MCF7 and MCF7F cells also reduced expression of the mitochondrial protective proteins MCL-1 and BCL-XL (); these are proteins we have previously implicated in breast cancer cells to sequester Beclin1 and reduce the ability of tumor cells to induce autophagy, as well as to suppress apoptosis. Over-expression of BCL-XL or expression of dominant negative caspase 9, but not expression of c-FLIP-s; significantly suppressed drug combination cytotoxicity, implying that autophagy was feeding in to the intrinsic apoptosis pathway at the level of the mitochondrion and that the extrinsic pathway was not involved in killing ().
Based on our in vitro cell survival data and expression data for autophagy regulatory proteins, we next examined the relative phosphorylation and expression levels of signal transduction proteins after pemetrexed + sorafenib treatment; proteins whose expression and activity has been associated by others and ourselves in a variety of systems to correlate to altered levels of autophagy and apoptosis. Tumor cell types that displayed high levels of cell killing after pemetrexed + sorafenib exposure, such as MCF7F, H460 and HuH7 tended to exhibit significantly elevated basal levels of AKT, p70 S6K and/or mTOR phosphorylation (; Figure S10
Knock down of PDGFRβ, mTOR or p70 S6K enhances pemetrexed toxicity and inhibition of ERK1/2 suppresses drug combination toxicity
Cells that were more sensitive to the pemetrexed + sorafenib drug combination, such as MCF7F and HuH7 also tended to display elevated expression levels of Class III RTKs such as PDGFRβ and VEGFR1; known in vivo targets of sorafenib. Thus, based on these findings, and those in with estrogen-dependent and fulvestrant-resistant MCF7 cells, we further explored the signaling responses and viability changes of tumor cells after pemetrexed + sorafenib treatment.
We found that fulvestrant resistant MCF7 breast cancer cells (MCF7F) over-expressed the class III RTK PDGFRβ, had elevated levels of ERK1/2, p70 S6K and mTOR activity, and were more sensitive to drug combination toxicity compared to their estrogen dependent counterparts (, ). Treatment of parental MCF7 cells with pemetrexed and sorafenib, but not the individual drugs, modestly suppressed T421/S424 and mTOR S2448 phosphorylation and strongly increased ERK1/2 phosphorylation (). Treatment of MCF7F cells with pemetrexed more effectively reduced p70 S6K T421/S424 phosphorylation than sorafenib, and sorafenib more effectively reduced p70 S6K T389 and mTOR S2448 phosphorylation than pemetrexed. Knock down of PDGFRβ, or of p70 S6K or mTOR enhanced pemetrexed and pemetrexed + sorafenib lethality (). Thus for PDGFRβ, the observed elevated receptor expression and elevated drug toxicity in MCF7F cells, define this protein as one molecular marker for a tumor cell response to the sorafenib and pemetrexed drug combination. Because sorafenib and pemetrexed treatment reduced p70 S6K and mTOR activity, we hypothesized that expression of constitutively activated forms of p70 S6K and mTOR would reduce drug cytotoxicity. Expression of a constitutively active form of p70 S6K and of mTOR significantly reduced the toxic effects of pemetrexed and sorafenib treatment ().
All the prior studies in the manuscript have been using 2D in vitro cultures of tumor cells. As our ultimate goal is to translate the pemetrexed + sorafenib drug combination into the clinic, we determined whether sorafenib and pemetrexed interacted in vivo to suppress tumor cell growth in various tumor model systems. In orthotopic established HER2 positive BT474 human mammary carcinoma tumors growing in the 4th mammary fat pad, sorafenib significantly reduced tumor growth whereas pemetrexed had little impact on tumor mass (). Combined exposure to sorafenib and pemetrexed significantly reduced tumor growth below that of sorafenib treatment alone and almost abolished tumor growth. The alterations in tumor growth data correlated in sections of the respective tumors with increased cleavage of pro-caspase 3 and TUNEL staining, a reduction in proliferation (Ki67) and a manifest disruption of tumor cyto-architecture ().
Pemetrexed and sorafenib interact to suppress breast cancer tumor growth in orthotopic human and rodent syngeneic model systems
In the spontaneous mouse mammary tumor cell line 4T1, that is HER1 dependent for growth, we noted in tumors growing in the 4th
mammary fat pad that sorafenib and pemetrexed also interacted to suppress tumor growth (). Our in vivo data using 4T1 cells was in agreement with in vitro apoptosis data in Figure S5
. In an orthotopic model of human GBM, in a primary GBM tumor cell isolate that displays invasive capabilities compared to commercially available established cell lines i.e. GBM6-luc that expresses EGFR vIII, we noted that treatment with pemetrexed + sorafenib significantly suppressed tumor cell growth during and shortly following drug exposure (Figure S11
, ; p < 0.05). Many days after drug exposure, tumors still exhibited a high level of apoptosis (Figure S12
). Finally, as we wish to move our drug combination into the clinic, consideration of normal tissue toxicity effects needed to be made. Thus we determined whether combined sorafenib and pemetrexed treatment had any deleterious effects on normal mouse tissues. Pemetrexed + sorafenib treatment did not reduce the body mass of animals carrying GBM6-luc, BT474 tumors or in animals lacking tumors (Figures S13–S15
) Two weeks of pemetrexed + sorafenib treatment did not result in any obvious normal tissue toxicity as judged using H&E staining of sectioned organs and examination of nuclear morphology (apoptosis) or of tissue integrity (Figures S16–S18