Tetrac and NDAT Induce Anti-proliferation via Integrin αvβ3 in Colorectal Cancers With Different K-RAS Status

Colorectal cancer is a serious medical problem in Taiwan. New, effective therapeutic approaches are needed. The selection of promising anticancer drugs and the transition from pre-clinical investigations to clinical trials are often challenging. The deaminated thyroid hormone analog (tetraiodothyroacetic acid, tetrac) and its nanoparticulate analog (NDAT) have been shown to have anti-proliferative activity in vitro and in xenograft model of different neoplasms, including colorectal cancers. However, mechanisms involved in tetrac- and NDAT-induced anti-proliferation in colorectal cancers are incompletely understood. We have investigated possible mechanisms of tetrac and NDAT action in colorectal cancer cells, using a perfusion bellows cell culture system that allows efficient, large-scale screening for mechanisms of drug actions on tumor cells. Although integrin αvβ3 in K-RAS wild type colorectal cancer HT-29 cells was far less than that in K-RAS mutant HCT116 cells, HT-29 was more sensitive to both tetrac and NDAT. Results also indicate that both tetrac and NDAT bind to tumor cell surface integrin αvβ3, and the agents may have different mechanisms of anti-proliferation in colorectal cancer cells. K-RAS status appears to play an important role in drug resistance that may be encountered in treatment with this drug combination.

Tetrac has been shown to compete with thyroid hormone, e.g., L-thyroxine (T 4 ) for the iodothyronine receptor on integrin αvβ3. Tetrac inhibits binding of thyroid hormones, thus inhibiting the downstream signal transduction pathways of nongenomically initiated effects of thyroid hormone (4,7,(19)(20)(21). Tetrac also has actions at the receptor that are independent of the functions of thyroid hormone, for example, modulation of angiogenesis by multiple mechanisms and regulation of tumor cell metabolism (22). Its interaction with integrin αvβ3 permits tetrac to modify differentially regulated gene expression that is related to cancer cell survival pathways. Tetrac upregulates expression of pro-apoptotic BcL-x short form (12), antiangiogenic thrombospondin 1 (THBS1) and other pro-apoptotic genes (4). In addition, tetrac downregulates transcription of several families of anti-apoptotic genes. A nano-formulation of tetrac, nano-diamino-tetrac (NDAT), has been shown to act primarily at the cell surface and does not enter the nucleus when it does enter the cell.
Recently we have studied the anti-proliferative effects of tetrac, NDAT, and their combinations with other anticancer drugs on colorectal cancer cells (4,7,13,(19)(20)(21). In combination with resveratrol, NDAT downregulated resveratrol-induced ribonucleotide reductase regulatory subunit M2 (RRM2) gene expression in vivo and potentiated the anticancer effect of the stilbene (20). In addition, tetrac enhanced nuclear abundance of chibby family member 1 (CBY1), a nuclear β-catenin antagonist, which is a protein that may compromise nuclear β-catenindependent gene expression and proliferation (6). Gefitinibinduced anti-proliferation in gefitinib-resistant colorectal cancer cells is restored by NDAT; the mechanism involves inhibition of beta-galactoside alpha-2, 6-sialyltransferase 1 (ST6Gal1) activity and PI3K activation (7). These observations indicate that added or enhanced effects are obtained with combinations of tetrac or NDAT and other chemotherapeutic agents.
In the current report, we investigated mechanisms by which tetrac-and NDAT induced anti-proliferation in colorectal cancer cells. In addition, we report studies conducted to define the different gene profiles induced by tetrac and NDAT in colorectal cancer cell lines. Finally, using a novel perfusion bellows cell culture system, we have distinguished the mechanisms by which tetrac or NDAT work on human colorectal cancer cells with different K-RAS status.

Pharmacodynamics
Anti-proliferative effects of tetrac and NDAT were defined in a well-established perfusion bellows cell culture system (13,23). At the outset, 5 × 10 7 cells were seeded in perfusion bellows cell culture system and incubated at 37 • C overnight. Then polymer flakes were harvested, trypsinized, and cells were collected and counted. The number of original cells attached to flakes was 0.5 × 10 7 cells/bottle. Cell cultures were refreshed with 1% stripped FBS-containing medium. Tetrac or NDAT was added in a medium bottle to the final concentrations indicated in the Results section. Specific concentration of tetrac and NDAT were chosen according to the physiological concentration of T 4 (10 −7 M) as described previously (24)(25)(26). The samples of cell-bearing flakes were then treated as indicated, and cells were harvested at timeframe indicated, trypsinized, and collected for counting.

Western Blotting
Western blotting analyses were conducted followed routine protocol described previously (6,7,(19)(20)(21)23). Consistent amount of protein samples were resolved on a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). A 20 µg quantity of protein was loaded in each well with 5x sample buffer, and the protein samples were resolved with electrophoresis at 100 V for 2 h. The resolved proteins were transferred from the polyacrylamide gel to Millipore Immobilon-PSQ Transfer PVDF membranes (Millipore, Billerica, MA, USA) with the Mini Trans-Blot R Cell (Bio-Rad Laboratories). The membranes were blocked with a solution of 2% FBS in Tris-buffered saline and incubated with primary antibodies to pERK1/2 (GeneTex, Inc., Hsinchu, Taiwan), Cyclin D1 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA), ERK1/2 (Santa Cruz Biotechnology), and α-Tubulin (Novus Biologicals, Littleton, CO, USA) at 4 • C overnight and washed; the proteins were detected with HRP-conjugated secondary antibodies and Immobilon TM Western HRP Substrate Luminol Reagent (Millipore). Images of the western blots were visualized and recorded with an Amersham Imager 600 (GE Healthcare, Chicago, IL, USA). The blots were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Data Analysis and Statistics
Data are presented as the mean ± S.E. The data were analyzed using IBM SPSS Statistics software version 19.0 (SPSS, Inc., Chicago, IL, USA). One-way analysis of variance (ANOVA) with Duncan's post-hoc test was used to analyze the differences between experimental groups followed by a paired Student's t-test. Student's t-tests for paired data were also used in some cases as indicated. p < 0.05 was considered statistically significant.

Different Expression Levels of Integrin αvβ3 Are Present on the Cell Surface of Colorectal Cancer Cells
To evaluate the expression of integrin αvβ3 in the colorectal cancer cell lines HCT116 and HT-29, studies of QPCR and flow cytometry analysis of integrin αvβ3 were conducted. The expressions of ITGαV and ITGβ3 in HCT116 cells were significantly higher than that in HT-29 cells (2.7 fold in ITGαV and 9.4 fold in ITGβ3) ( Figure 1A, left panel). The expression of integrin αvβ3 on the cell surface of HCT116 cells (35.6%) was also notably greater than that on HT-29 cells (12.6%) ( Figure 1A, right panel).

Both Tetrac and NDAT Bind to Cell Surface Integrin αvβ3 in Colorectal Cancer Cells
To determine whether the signal transduction pathways activated by tetrac and NDAT are integrin αvβ3-dependent, colorectal (Data are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, compared with untreated control; # p < 0.05, ### p < 0.001, compared with anti-integrin αvβ3 antibody treatment. a-c: the subsets after post hoc analysis after the significant differences were obtained using one-way ANOVA).
cancer K-RAS-mutant HCT116 cells and K-RAS-wild type HT-29 cells were set in 10 mL Petri dishes and starved with serumfree media for 2 days. Prior to starting each experiment, cells were re-fed with fresh medium. Cells were treated with 1 µg/mL anti-integrin αvβ3 30 min before treatment of either 10 −7 M tetrac or NDAT for 30 min. Total proteins were extracted and Western blot analyses were conducted for pERK1/2 study. Tetrac inhibited activation of ERK1/2, but NDAT increased activated ERK1/2 in HT-29 cells (Figure 1B).
On the other hand, tetrac and NDAT inhibited constitutively activated ERK1/2 in HCT116 cells ( Figure 1C). However, this effect was partially removed by anti-integrin αvβ3 antibody pretreatment (Figures 1B,C). Parallel studies were conducted to define the expression of specific genes controlled by tetrac or NDAT. Colorectal cancer HCT116 cells were set in 6-well trays and starved with serumfree media for 2 days. Prior to starting each experiment, cells were re-fed with fresh medium. Cells were treated with 1 µg/mL anti-integrin αvβ3 for 30 min prior to treatment with 10 −7 M tetrac or NDAT for 24 h. Total RNA was extracted and qPCR was conducted for integrin αv (ITG αv), integrin β3 (ITG β3), CASP2, CCND1, c-Myc, and THBS1. Results shown in Figure 2 indicate that both tetrac and NDAT inhibited expression of CCND1 and c-Myc, but promoted expression of CASP2 and THBS1. The effect of NDAT was much greater than that of tetrac. However, pretreatment of cells with anti-integrin αvβ3 blocked the effect of tetrac and NDAT (Figure 2). These results suggest that both tetrac and NDAT bind to integrin αvβ3 to induce downstream signals that play an important role in tetrac/NDAT-dependent cellular activities.
Cells treated with tetrac and NDAT were also prepared for flow cytometry studies. Results shown in Figure 5 indicate that tetrac induced cancer cell arrested in G2/M stage. However, low concentration 10 −8 M NDAT induced G2/M arrest and 10 −7 M NDAT shut down DNA synthesis.

NDAT and Tetrac Modulate Expression of Different Genes in Colorectal Cancer Cells
We observed previously that treatment with tetrac or NDAT results in different gene expression profiles in MDA-MB-231 cells and in medullary thyroid carcinoma cells (24). In order to define the gene transcription profiles induced by tetrac and by NDAT in colorectal cancer cells with different K-RAS status, HT-29 cells and HCT116 cells were treated with 10 −8 and 10 −7 M tetrac or NDAT, respectively for 24 h. RNA was extracted from the harvested cells at the end of treatment for qPCR studies. Treatment with NDAT (10 −8 M) for 24 h increased expression of p53 and p53-responsible genes such as p21, PIG3 (TP53I3), BAD, and CASP2 in HT-29 cells significantly (Figure 5). In addition, NDAT increased expression of THBS1. Protein TSP1 is an endogenous suppressor of angiogenesis and is invariably suppressed in cancer cells. Although tetrac also increased expression of those genes, the effect was 10-fold less (Figure 5). In contrast, tetrac (10 −8 and 10 −7 M) significantly increased expression of p21, p53, PIG3, BAD, and THBS1 genes in HCT116 cells (Figure 6). However, 10 −8 M tetrac did not significantly inhibit expression of VEGF-A, which plays an important role in HCT116 cells metastasis (Figure 6). On the other hand, NDAT also increased expression of the mentioned genes and significantly decreased expression of VEGF at 10 −8 M.

DISCUSSION
The perfusion bellows cell culture studies we describe here provide useful pharmacodynamic information of the application HT-29 colorectal cancer cells were grown in the perfusion bellows system and treated with tetrac (10 −8 to 10 −6 M) or NDAT (10 −9 to 10 −7 M) for 6 days. After incubated in the treatment, cells were isolated and subjected to analysis by cell count, N = 3. Data are expressed as mean ± SD; ***p < 0.001, compared with untreated control. a-c: the subsets after post hoc analysis after the significant differences were obtained using one-way ANOVA.
of new drugs or combinations of various agents in vitro to human cancer cell lines (13). Pharmacodynamic modeling based FIGURE 4 | Tetrac (A) and NDAT (B) induce anti-proliferation of K-RAS-mutant colorectal cancer HCT116 cells. HCT116 colorectal cancer cells were grown in the perfusion bellows system and treated with tetrac (10 −8 to 10 −6 M) or NDAT (10 −9 to 10 −7 M) for 6 days. After incubated in the treatment, cells were isolated and subjected to analysis by cell count. N = 3. Data are expressed as mean ± SD; ***p < 0.001, compared with untreated control. a-c: the subsets after post hoc analysis after the significant differences were obtained using one-way ANOVA.
on the expected pharmacokinetics of a drug permits-in the perfusion bellows cell culture system-the understanding of doseresponse relationship of antineoplastic agents over a very wide range of concentration in vitro and can support translation from in vitro models to animal models and ultimately human clinical trials.
Via binding to the thyroid hormone receptor on plasma membrane integrin αvβ3 (27,28), NDAT induces signal transduction and biological activities similar to those of unmodified tetrac (Figures 1, 2), but with desirable effects on cell survival pathway genes that differ from the parent thyroid hormone analog (Figures 2, 5, 6) (5,24). Studies indicated that NDAT but not tetrac activated ERK1/2 in K-RAS-wild type colorectal cancer HT-29 cells (Figure 1A), which confirms previous results that NDAT activates ERK1/2 in HT-29 cells (24). On the other hand, both tetrac and NDAT inhibited ERK1/2 activation in K-RAS-mutant HCT-116 cells (Figure 1B). Interestingly, NDAT is more sensitive to the inhibitory effect of pretreatment of cells with of anti-integrin αvβ3 (Figure 2). Both tetrac and NDAT induced anti-proliferation in colorectal cancer K-RAS-wild type HT-29 cells and in K-RAS-mutant HCT116 cells (Figures 3, 4). The growth-inhibitory effects of tetrac and NDAT on HCT116 colon cancer cell xenografts are evident in 2-4 d after the onset of treatment (6,7,20). The anti-proliferative effect of tetrac and NDAT on cancer cells in the perfusion bellows cell culture system was seen at 3 d after the start of treatment (Figures 3, 4). Pharmacodynamics of anti-proliferative activities in vitro of tetrac and NDAT in colorectal cancer cells were compared in the perfusion bellows cell culture system. Results revealed that NDAT had a higher potency than tetrac as an antiproliferative agent (Figure 3). We have previously shown that the anticancer effects of tetrac and NDAT in colorectal cancer HCT116 cell xenografts are well-established within 3 d after onset of drug administration (6,7). Although inhibitory effects of tetrac and NDAT in the perfusion bellows cell culture system are limited to suppression of cell proliferation, these results in the perfusion system reproduce findings obtained earlier in the same cells in xenografts. Tetrac-/NDAT-induced anti-tumor effects in xenografts have been shown to involve both primary effects on tumor cell proliferation and anti-angiogenesis effect (6,7). Results in these sets of studies similarly demonstrate that the anticancer effects of tetrac/NDAT occur first as anticancer cell proliferation.
The inhibition of HT-29 cell growth by NDAT and tetrac was comparable at a higher drug concentration (10 −7 M), but there was increased sensitivity to NDAT at a lower drug concentration of the used agents (Figure 3). In addition to the anti-proliferative effects initiated at the cell surface integrin receptor, tetrac may penetrate into cells to exert low-grade thyromimetic (proliferative) effects in the nucleus of treated colorectal cancer cells. Therefore, the net anti-proliferative effect of tetrac decreases. On the other hand, NDAT does not gain access to the cell nucleus and shows a more robust antiproliferative effect.
In addition to their anti-proliferative effects on cancer cells, tetrac and NDAT have been shown to enhance anti-cancer growth by other anticancer drugs. Cetuximab (Erbitux R ) inhibits K-RAS wild-type but not K-RAS mutant colorectal cancer cell growth (19). The combination of tetrac and cetuximab significantly reduced cell proliferation compared to cetuximab FIGURE 5 | Tetrac and NDAT suppress expression of proliferative and metastasis-related genes in HT-29 cancer cells. HT-29 cells were seeded in 6-well plates and treated with different concentrations of 10 −8 and 10 −7 M tetrac or NDAT for 24 h. Cells were harvested and total RNA was extracted. qPCR experiments were conducted to examine expression of anti-proliferative genes (p21, p53, and PIG3), apoptotic genes (BAD and CASP2) and metastasis-related genes (THBS1). N = 6. (Data are expressed as mean. ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, compared with untreated control).
alone in K-RAS mutant HCT 116 cells, but not in K-RAS wild type COLO 205 cells (19). NDAT also rescued the antiproliferative effect of cetuximab in both colorectal cancer cell lines (19). These results suggest that tetrac and NDAT may be used in the clinic in the future to treat K-RASmutant colorectal cancer patient. However, present studies indicated that NDAT was much more effective than tetrac (Figures 5, 6). Furthermore, NDAT reversed K-RAS mutantdependent cetuximab resistance. In addition, xenograft weights in NDAT-treated alone animals are not significantly decreased compared to those in untreated control [result not shown (6,7)]. Therefore, NDAT alone or combination with low dosage of cetuximab may be a new chemotherapeutic approach in the future.
Studies have shown that tetrac affects only XIAP gene expression (12) in breast cancer cells. On the other hand, NDAT downregulates expression of apoptosis inhibitors XIAP, myeloid cell leukemia sequence 1 (MCL1), and apoptosispromoting genes such as CASP2 and BCL2L14 (12). Expression of the angiogenesis inhibitor thrombospondin 1 (THBS1) gene is increased by both unmodified tetrac and NDAT, as is the expression of CBY1, a nuclear inhibitor of catenin activity (12). The majority of differentially regulated K-RAS-oncogene family members and the epidermal growth factor receptor gene are downregulated by NDAT, but not by tetrac (12). β-Catenin belongs to a class of catenins that have been shown to play roles in cell-cell adhesion. It also has transcription functions. Various cancers such as those of the colorectum, FIGURE 6 | Tetrac and NDAT suppress expression of proliferative and metastasis-related genes in HCT116 cancer cells. HCT116 cells were seeded in 6-well plates and treated with different concentrations of 10 −8 and 10 −7 M tetrac or NDAT for 24 h. Cells were harvested and total RNA was extracted. Studies of qPCR were conducted to examine expression of anti-proliferative genes (p21, p53, and PIG3), apoptotic gene (BAD), and metastasis-related genes (VEGF-A and THBS1). N = 6. (Data are expressed as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001, compared with untreated control).
breast, and ovary have been shown to contain mutant and overexpressed β-catenin genes. CBY1, an inhibitor of nuclear functions of β-catenin, is upregulated by NDAT. Like β-catenins, integrin αvβ3 participates in cellular adhesion complexes. NDAT downregulates expression of the gene for integrin β3 monomer. Since integrin αvβ3 has been shown to involve metastasis and migration, this may be a desirable action of NDAT at αvβ3 in cancer cells.
NDAT also downregulates expression of the genes for α-catenins, CTNNA1 and CTNNA2 (8). Mutation of CTNNA2 is associated with tumor invasiveness and thus inhibition of transcription of the gene is desirable. The nonmutated gene product of CTNNA1 can function as a tumor invasion suppressor, but mutation is associated with gastrointestinal tract and other cancers.
Thyroid hormone induces expression of MMP-2 and MMP-9 in myeloma cells and colorectal cancer cells (7). This action is inhibited by tetrac and NDAT; this implies that αvβ3 can be involved in the contribution of matrix metalloproteinases to cancer-relevant angiogenesis and to tumor invasiveness. Tetrac and NDAT were able to inhibit constitutive expression of VEGF-A (Figure 6). Tetrac and NDAT also increased transcription of thrombospondin 1 (THBS1, TSP1), which suppresses angiogenesis.
In summary, tetrac and NDAT via integrin αvβ3 regulate thyroid hormone-dependence of various independent cancer cell activities. NDAT is much more potent than tetrac and has a broader range of transcriptional actions in cancer cells that may be clinically desirable.