Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer - PubMed (original) (raw)
Thioredoxin interacting protein (TXNIP) is a novel tumor suppressor in thyroid cancer
Jennifer A Morrison et al. Mol Cancer. 2014.
Abstract
Background: Thyroid cancer is the most common endocrine malignancy, and many patients with metastatic differentiated thyroid cancer (DTC), poorly differentiated thyroid cancer (PDTC), and anaplastic thyroid cancer (ATC) fail to respond to conventional therapies, resulting in morbidity and mortality. Additional therapeutic targets and treatment options are needed for these patients. We recently reported that peroxisome proliferator-activated receptor gamma (PPARγ) is highly expressed in ATC and confers an aggressive phenotype when overexpressed in DTC cells.
Methods: Microarray analysis was used to identify downstream targets of PPARγ in ATC cells. Western blot analysis and immunohistochemistry (IHC) were used to assess thioredoxin interacting protein (TXNIP) expression in thyroid cancer cell lines and primary tumor specimens. Retroviral transduction was used to generate ATC cell lines that overexpress TXNIP, and assays that assess glucose uptake, viable cell proliferation, and invasion were used to characterize the in vitro properties of these cells. An orthotopic thyroid cancer mouse model was used to assess the effect of TXNIP overexpression in ATC cell lines in vivo.
Results: Using microarray analysis, we show that TXNIP is highly upregulated when PPARγ is depleted from ATC cells. Using Western blot analysis and IHC, we show that DTC and ATC cells exhibit differential TXNIP expression patterns. DTC cell lines and patient tumors have high TXNIP expression in contrast to low or absent expression in ATC cell lines and tumors. Overexpression of TXNIP decreases the growth of HTh74 cells compared to vector controls and inhibits glucose uptake in the ATC cell lines HTh74 and T238. Importantly, TXNIP overexpression in T238 cells results in attenuated tumor growth and decreased metastasis in an orthotopic thyroid cancer mouse model.
Conclusions: Our findings indicate that TXNIP functions as a tumor suppressor in thyroid cells, and its downregulation is likely important in the transition from differentiated to advanced thyroid cancer. These studies underscore the potential of TXNIP as a novel therapeutic target and prognostic indicator in advanced thyroid cancer.
Figures
Figure 1
TXNIP is highly expressed in DTC cell lines and low or undetectable in ATC cell lines, and glucose uptake is inversely proportional to TXNIP expression levels. (A) HTh74 cells were transduced with lentivirus expressing a PPARγ-specific shRNA or scrambled control, and stable pools were generated under antibiotic selection. Western blot analysis of nuclear lysates (top immunoblot) reveals PPARγ expression levels in the transduced cells. Using Western blot analysis of whole cell lysates, TXNIP and β-actin (loading control) were detected using specific antibodies. Nonspecific band is indicated by “ns”. (B) Western blot analysis for TXNIP, Trx-1, and β-actin (loading control) were performed on whole cell lysates prepared from a panel of DTC and ATC cell lines grown under standard conditions. (C) Glucose uptake assays were performed. Each condition was performed in triplicate per experiment, and each experiment was performed at least three times. Nonspecific glucose uptake as determined by parallel treatment of a subset with cytochalasin B was subtracted from measurements. Data from all experiments were combined, and glucose uptake from each cell line was normalized to levels of HTh74 (average set at 1). Normalized averages are plotted, and error bars indicate SEM. ***p <0.0001, **p <0.001.
Figure 2
TXNIP is expressed in primary DTC tumors but is low or undetectable in ATC tumors. Immunohistochemistry was performed using a TXNIP-specific primary antibody or normal mouse IgG control and a horseradish peroxidase tagged secondary antibody on a panel of 13 PTC and 8 ATC patient specimens. Two representative PTC (A-B) and one ATC (C) specimen stains are shown at 20X and 60X power. Positive staining is indicated by brown coloring, though staining of colloid is nonspecific. Staining was quantitated by a pathologist (S. B. S.), and intensity of staining was scored from absent (0) to high to (3+), followed by percent of tumor positivity.
Figure 3
TXNIP overexpression in ATC HTh74 and T238 cells attenuates glucose uptake. HTh74 and T238 cells were transduced with retrovirus encoding human TXNIP or vector control as well as a selectable antibiotic resistance marker, and stable pools were generated under antibiotic selection. Western blot analysis of whole cell lysates with TXNIP- and β-actin-specific antibodies is shown for HTh74 (A) and T238 (B). Glucose uptake assays were performed as described in Figure 1 using the HTh74 stable cell lines (C) and T238 stable cell lines (D). Data from all experiments were combined, and glucose uptake from each cell line was normalized to vector control levels (average set at 1). **p = 0.001, ***p <0.0001.
Figure 4
TXNIP overexpression inhibits in vitro growth of ATC HTh74 cells. Viable cell proliferation assays were performed for stable HTh74 (A) and T238 (B) cell lines. Briefly, 50,000 cells were plated in 6-cm plates, trypsinized and viable cell counts were determined using the ViCell automated cell counting system. Each time point was performed in duplicate, and lysates were prepared from the day 7 time point to confirm high TXNIP expression using Western blot analysis. Each experiment was performed at least three times. Mean and SEM are plotted. Closed squares (∎) indicate vector control cells, and open diamonds (◊) indicate cells with TXNIP overexpression. In vitro invasion assays were performed on the TXNIP-overexpressing stable HTh74 (C) and T238 (D) cell lines as described in the Methods section. Results from three independent experiments were combined and normalized to the vector control average and graphed with mean plus SEM. *p <0.01 (Figure 4A), Figure 4B p = 0.9021, Figure 4C p = 0.3523, Figure 4D p = 0.0754.
Figure 5
TXNIP overexpression in ATC T238 cells attenuates tumor growth and metastasis in an in vivo orthotopic murine thyroid cancer model. T238 cells stably expressing luciferase-IRES-GFP and either TXNIP or empty vector were injected into the right lobe of the thyroid gland of athymic nude mice with the aid of a dissecting microscope to enhance visualization. Weekly imaging with IVIS after injection of luciferin was performed to monitor tumor establishment and growth. There were 10–11 mice per group in each experiment, and the experiment was performed two times. (A) Representative images of one mouse per group imaged by IVIS over time are shown. (B) Quantitation of the bioluminescence from one experiment is shown with average and SEM. Closed squares (∎) indicate vector control group, and open diamonds (◊) indicate TXNIP group. (C) Final tumor volumes, as calculated from caliper measurements, from both experiments are combined and plotted with averages and SEM. To assess for lung metastasis, RNA isolated from whole lungs was subjected to qRT-PCR analysis to assess GFP expression. Data was normalized to 18 s RNA levels, and averages plus SD were plotted (D). RNA isolated from lungs of mice receiving no tumor cell injections was included as an additional negative control for GFP expression and is indicated with closed circles (●). *p <0.05, **p =0.0021, ***p <0.001, ****p <0.0001.
References
- Parikh H, Carlsson E, Chutkow WA, Johansson LE, Storgaard H, Poulsen P, Saxena R, Ladd C, Schulze PC, Mazzini MJ, Jensen CB, Krook A, Bjornholm M, Tonqvist H, Zierath JR, Ridderstrale M, Altshuler D, Lee RT, Vaag A, Groop LC, Mootha VK. TXNIP regulates peripheral glucose metabolism in humans. PLoS Med. 2007;4:e158. doi: 10.1371/journal.pmed.0040158. - DOI - PMC - PubMed
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