2.2. Treatment with Vemurafenib and Temsirolimus Induces Anti-Proliferative Effects in H1_DL2 and H3 Cell Lines Grown as Monolayers
The H1_DL2 cell line was effectively treated with vemurafenib, with an IC50
of 0.679 μM (Figure 1A
, left). Treatment with temsirolimus alone was less effective, with an IC50
of 4.323 μM (Figure 1A
, middle), while combined therapy was shown to be the most effective treatment (IC50
= 0.063 μM; Figure 1A
A more detailed comparison of the drug effects on the H1_DL2 cells is shown in Figure 1B
and Table 1
. At a drug concentration of 0.05 μM, 82.8% of the H1_DL2 cells survived treatment when using vemurafenib, while 54.7% of the cells survived treatment with temsirolimus alone. However, only 31.0% of the cells survived a combined treatment, indicating a synergistic effect of combined therapy (co-efficient of drug interaction (CDI), 0.68; see Table 1
). Single drug treatment with vemurafenib was effective at concentrations of 5 or 10 μM (29.5% and 24.4% survival, respectively), while treatment with temsirolimus showed a cell survival of 53.1% (5 μM) and 48.6% (10 μM). Combined treatment was the most effective, with cell survival of 21.0% at 5 μM (synergistic effect) and 15.9% at 10 μM. Pictures of cell survival after treatment with vemurafenib and temsirolimus are also seen in Figure S2. For a comparison, we assessed the treatment effects on the H3 melanoma brain metastasis cell line, which expresses WT BRAF and has a homozygous deletion of PTEN (data not shown). In general, the H3 cells were not as sensitive to therapy as the H1_DL2 cells, where vemurafenib treatment resulted in an IC50
of 5.105 μM (Figure 1C
, left). Treatment with temsirolimus alone was even less effective, with an IC50
of 9.906 μM (Figure 1C
, middle). An increased effect using a combined therapy was not seen (IC50
= 6.446 μM; Figure 1C
A more detailed comparison of the drug effects on the H3 cells is shown in Figure 1D
and Table 2
. All H3 cells survived treatment with 0.05 of μM vemurafenib, while 87.6% of the cells survived treatment with temsirolimus alone.
The combined treatment did not increase cell death, as 85.6% of the cells survived treatment. At 5 μM, 64.0% of the cells survived after vemurafenib treatment, while 79.0% of the cells survived when temsirolimus was used alone. Combined therapy at this dose was not more effective (75.6% survival). Similar results were observed at 10 μM (42.3% survival with vemurafenib, 79.8% survival using temsirolimus and 46.8% survival after combined treatment).
Melanoma patients with advanced disease have a high chance of developing brain metastases, which is associated with a poor prognosis. Apart from introducing Gamma Knife radiosurgery, conventional treatment of these metastases has not changed during the last two decades [3
], necessitating the development of new therapeutic strategies. Several small molecule inhibitors targeting signaling pathways have been developed recently. However, with a few exceptions [24
], they have not been studied clinically on patients with brain metastases from melanoma.
The two specific protein kinase inhibitors, vemurafenib [34
] and temsirolimus [2
], have previously been shown to target the MAPK and PI3K pathways, respectively. Lazar and colleagues showed that more than 10% of human melanomas display a loss of PTEN expression, in addition to BRAFV600E
]. PTEN is one of the critical negative regulators of AKT activity [40
]. Thus, the loss of PTEN expression leads to increased PI3K/AKT signaling, particularly when BRAF is inhibited, which, in turn, may contribute to an intrinsic resistance of BRAFV600E
mutated melanoma cells to vemurafenib treatment [17
]. Thus, it is clear that single drug therapy targeting individual pathways may be insufficient to obtain therapeutic efficacy. The fact that melanomas develop resistance to vemurafenib emphasizes the importance of targeting multiple pathways simultaneously [9
We set out to study whether the growth and proliferation of human melanoma brain metastasis cells could be inhibited more efficiently in vitro by combining therapies targeting two major pathways in melanoma progression. To our knowledge, this study is the first to demonstrate inhibitory effects on melanoma brain metastatic cell lines by combining vemurafenib and temsirolimus, and we show the regulation of genes after treatment that may be involved in reducing the invasive potential of melanoma cells.
The BBB has an important role in the treatment of brain metastasis. The delivery of chemotherapy is usually ineffective, due to an often intact vascular barrier in the brain with low passive transcellular permeability. Furthermore, the endothelial cells of the brain vasculature express active efflux drug transporters, which limit the uptake of most anticancer drugs. We have previously shown in an animal model of brain metastasis that the BBB is relatively leaky for small contrast agents (molecular weight (MW) below 300 Da) at early stages and permeable to magnetic resonance imaging (MRI) contrast agents (MW 0.566 and 2.066 kDa) at later stages of brain metastatic development [4
]. These results suggest that small MW drugs, such as vemurafenib and temsirolimus, could potentially cross the BBB early in the development of brain metastases.
By targeting BRAF and mTOR simultaneously, we were able to inhibit the cell growth and proliferation (Figure 1
) of the H1_DL2 cells in monolayers. Combined treatment resulted in a synergistic increase in cell death compared to single drug treatment (at concentrations of 0.05 and 5 μM), as shown by the resazurin assay (Table 1
). However, temsirolimus alone was not able to decrease the migratory capacity of cells grown as monolayer cultures, compared to untreated cells. These results were supported by our gene analysis data, indicating minimal effects on genes related to invasive capacity after single dose treatment with temsirolimus (Figure 7B
; Group 9). The gene analysis, however, indicated that combined therapy was more effective at inhibiting cell invasion, compared to single-dose treatment with vemurafenib.
Assays using tumor cells grown as multicellular spheroids are regarded as more relevant with respect to solid tumor growth seen in vivo
]. In our study, vemurafenib was more effective than temsirolimus in inhibiting spheroid growth in vitro
, and the combined therapy suggested an additional growth inhibition. The number of dead cells within the spheroids did not increase after combined treatment compared to vemurafenib treatment alone, which likely can be explained by the previously observed poor penetrance of ethidium homodimer into compact spheroid structures. Our results on monolayer cells and spheroids, taken together, indicate that combined therapy may inhibit single tumor cell growth, as well as when treating solid tumors, provided that the drugs would effectively cross the BBB. To study this further, upcoming animal studies will investigate the effects of single and combined treatment on brain metastasis, both at the single-cell level in the animal brain using iron oxide prelabeling of the tumor cells [31
] and on solid tumor development.
The western blots showed that the expression of pMAPK was lost after combined treatment, and the expression of p-mTOR pAKT was reduced, suggesting that the MAPK and PI3K signaling pathways were inhibited.
Gene expression profiling is important for the identification of genes that may be involved in tumor progression and metastasis and also for studying how tumor cells respond to therapy. In addition, gene expression profiling has been important for a molecular sub-classification of tumors [42
]. PCA is an exploratory visualization of gene expression data and discriminates between sample groups [32
]. In our treatment study, PCA separated the sample groups for each respective treatment (Figure 5
) into tightly pooled clusters well.
A functional analysis of negatively or positively regulated genes before and after combination treatment was performed using IPA. Interestingly, pathways representing cell cycle regulation were downregulated after combined treatment of our melanoma brain metastasis cells (Figure 6
Further analysis of these pathways using the activation z
-score (Figure 7A
and Table 3
) showed seventeen downstream functions that were either activated (z
-score > 2) or inhibited (z
-score < −2) by combined treatment. These functions could be grouped into four different categories: (1) cell cycle; (2) cell death and cell survival; (3) cellular movement; and (4) DNA replication, DNA recombination and DNA repair. We then performed a more detailed analysis of the downstream functions with the highest relative z
-score, to study the differences in gene expression after single drug treatment and combined treatment. Gene sets related to G1/S phase, cell survival and cell invasion showed larger effects on gene expression after combined treatment, compared to the single drug treatments (Figure 7B
). Interestingly, vemurafenib treatment did not show any effects on downstream functions related to the apoptosis of melanoma cell lines, while DNA repair mechanisms resulted in low action scores (<−2) irrespective of treatment. Based on our functional analysis, further work is warranted to verify key genes that may represent therapeutic targets. This was, however, beyond the scope of this study.