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Combined MEK and Pi3'-kinase Inhibition Reveals Synergy in Targeting Thyroid Cancer in Vitro and in Vivo
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Combined MEK and Pi3'-kinase Inhibition Reveals Synergy in Targeting Thyroid Cancer in Vitro and in Vivo
Free PMC article
Anaplastic thyroid cancers and radioiodine resistant thyroid cancer are posing a major treat since surgery combined with Iodine131 therapy is ineffective on them. Small-molecule inhibitors are presenting a new hope for patients, but often lead to drug resistance in many cancers. Based on the major mutations found in thyroid cancer, we propose the combination of a MEK inhibitor and a Pi3'-kinase inhibitor in pre-clinical models. We used human thyroid cancer cell lines and genetically engineered double mutant BRAFV600E PIK3CAH1047R mice to evaluate the effect of both inhibitors separately or in combination in terms of proliferation and signaling in vitro; tumor burden, histology, cell death induction and tumor markers expression in vivo. The combination of MEK and Pi'3-kinase inhibition shows a synergistic effect in term of proliferation and apoptosis induction through Survivin down-regulation in vitro. We show for the first time the effects of the combination of a MEK inhibitor and Pi3'-kinase inhibitor in a genetically engineered mouse model of aggressively lethal thyroid cancer. In fine, the two drugs cooperate to promote tumor shrinkage by inducing a proliferation arrest and an elevation of apoptosis in vivo. Moreover, a phenotypic reversion is also observed with a partial restoration of normal thyroid marker transcription, and thyroid cancer marker expression reduction.In conclusion, combination therapy of MEK and Pi3'-kinase inhibition synergizes to target double mutant thyroid cancer in vitro and in vivo. This multidrug approach could readily be translated into clinical practice and bring new perspectives for the treatment of incurable thyroid carcinoma.
BRAF; Pi3K; combination treatment; genetically engineered mice; thyroid cancer.
Conflict of interest statement
CONFLICTS OF INTEREST
The author(s) declare(s) that they have no competing interests.
Figure 1. Drug combination synergistically inhibits thyroid cancer cell proliferation
A) Differential response to single and combination treatment with MEK and PI3′-kinase inhibitors: Human anaplastic thyroid cancer cells were treated with increasing concentrations of single drugs PD-325901, GDC-0941 or the combination at a constant ratio for about 3 doubling population times. All drug concentrations were normalized to IG50 equivalents of single agents (see Materials and Methods). Optical Density values were normalized to the untreated. All the measurements were done in triplicate and the mean was represented with SEM. The constant ratio approach allows figuring out obvious synergistic effect between drugs and calculating the combination index. ( B) Combination index (CI) as a function of Fraction affected (Fa): Combination index curves were calculated using Compusyn Software 2.0 based on Chou and Talalay method. The horizontal red line indicates the synergistic effect threshold. ( C) Cell cycle analysis on cells treated with single drug or the combination: Cell cycle analysis was performed on 8505c, OCUT-2 and SW1736 treated for 24 h with PD-325901 at 100 nM and/or GDC-0941 at 1 μM. Bars represent percentage of cells in each cell cycle stage as the mean of triplicates from independent experiments with the calculated Standard Deviation.
Figure 2. Drug combination synergistically induces apoptosis in anaplastic thyroid cancer cell lines
A) Flow cytometry analysis of OCUT-2 cells treated with 100 nM of PD-325901, 1 μM of GDC-0941 or the combination for 24 h stained with FITC annexinV and PI. Apoptosis was detected by FITC annexinV binding to the cells. Quadrant Lower Left, FITC annexinV(−) PI(−) represents living cells. Quadrant Lower Right, PI(+) represents cells undergoing necrosis. Quadrant Upper Right, FITC annexinV(+) PI(+) represents cells in the late apoptosis and undergoing secondary necrosis. Quadrant Upper Left, FITC annexinV (+) PI(−) are cells in early apoptosis. The histograms represent the quantifications of intact cells, early apoptotic cells, late apoptotic cells and necrotic cells in the 3 cell lines after 24 h treatments as the mean of triplicate from 3 independent experiments. Error bars represent SEM. ( B) Flow cytometry analysis of OCUT-2 cells treated with 100 nM of PD-325901, 1 μM of GDC-0941 or the combination for 48 h stained with FITC annexinV and PI. The histograms represent the quantifications of intact cells, early apoptotic cells, late apoptotic cells and necrotic cells in the 3 cell lines after 48 h treatments as the mean of triplicate from 3 independent experiments. Error bars represent SEM.
Figure 3. The combination PD-325901/GDC-0941 inhibits MAPK and Pi3′-kinase pathways and leads to caspase dependent cell death enhanced by Survivin downregulation
Western blot showing the expression of component of signaling pathways and proteins involved in apoptosis caspase dependent and caspase independent in the 3 ATC cell lines treated for 24 h with single treatments or the combination PD-325901/GDC-0941. The quadrants in red correspond to the nuclear fraction and in black to the total extract.
Figure 4. The combination is more beneficial after 9 weeks of treatment in terms of tumor burden reduction compared to PD alone and shows histology improvement
A) Thyroid tumor burden during treatment measured by ultrasound imaging expressed in percentage of the starting tumor burden. The mean of each treated group was calculated with SEM. 2-way ANOVA test with Tukey as post hoc test were used for multiple comparison ( B) Representative pictures of ultrasound pictures at the end of the treatment (9 weeks). ( C) Hematoxylin and Eosin stainings at 5× and 40× magnification of representative thyroid tissues after 9 weeks of treatments of mice by oral gavage with Vehicle, PD-325901 at 5 mg/kg, GDC-0941 at 50 mg/kg and combination.
Figure 5. PD and GDC drugs are hitting their target
in vivo based on the decreased phosphorylation of ERK and AKT
A) Western blot showing the expression of pERK1/2, pAKT, pERK, panAKT in the tumors of control and treated mice for 9 weeks ( B) Quantification of ratio pERK/totERK and pAKT/panAKT after 9 weeks of treatment. Means are represented with SEM and One-way Anova test with Dunnett as post hoc were used to assess statistical significance ( C) Western blot showing the expression of pERK1/2, pAKT, pERK, panAKT in the tumor of control and treated mice with 1 shot GDC-0941 at 50 mg/kg by oral gavage. ( D) Quantification of ratio pERK/totERK and pAKT/panAKT after short treatment with GDC-0941 (1× 50 mg/kg). the statistical significance was evaluated using a T test.
Figure 6. Tumors regrow after treatment release with different rate and seem to lose the histological improvement observed under treatment
A) Tumor burden during treatment (9 weeks) and regrowth after treatment removal up to 20 weeks measured on remaining mice. ( B) Hematoxylin and Eosin staining at 5× and 40× magnification of representative thyroids tissues after treatment removal with a control mouse euthanized at week 11 after reaching endpoint. PD-325901, GDC-0941 and combination treated mice were sacrificed at week 20 (11 weeks after drug release).
Figure 7. Drug treatments induce a decrease in proliferation and increased cell death
Representative immunofluorescence images of isolated thyroid tissues and corresponding quantification. The tissues were isolated after 9 weeks of treatment. (
A) Blue: DAPI and Green: Ki67. The graph shows the percentage of Ki67 positive cells. (B) Green: CC3. The graph displays the count of CC3 apoptotic bodies normalized to nuclei number. ( C) Green: TUNEL. The graph depicts the count of TUNEL positive apoptotic bodies normalized to the number of nuclei. All quantifications were performed on whole tumor sections using Quant Center software from 3DHISTECH. The mean of different measurements (thyroid lobes) are represented for each treatment group with SEM. One-way ANOVA test with Tukey as post hoc test were used to calculate statistical significance.
Figure 8. MEK inhibition alone or combined with Pi3′-kinase inhibition leads to a reduction in tumoral markers and an up-regulation of normal phenotype markers
A) Representative immunofluorescence images at 4×, 20× and 40× magnifications of isolated thyroid tissues from mouse treated 9 weeks with drugs. Blue: DAPI, Red: CK19, Green: Galectin-3. ( B) mRNA expression levels of proteins involved in the normal function of the thyroid normalized to the untreated expressed in fold induction. Actin was used as house keeping gene and all measurements were done in triplicate and represented as the mean with SEM. One-way ANOVA test and Tukey test were used for multiple comparison.
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