Abstract
Context:
Previously we identified RTN4IP1 to be differentially expressed in thyroid cancer by sex and the gene is located on chromosome 6q21, a chromosomal region frequently deleted or with loss of heterozygosity in a variety of human malignancies including thyroid cancer.
Objective:
Because the expression and function of this gene is unknown, we sought to characterize its expression in normal, hyperplastic, and benign and malignant thyroid tissue samples and to evaluate its function in cancer cells.
Design:
RTN4IP1 expression was analyzed in normal and hyperplastic thyroid tissue and benign and malignant thyroid tissue samples. In 3 thyroid cancer cell lines (TPC1 from a papillary thyroid cancer, FTC133 from a follicular thyroid cancer, XTC1 from a Hürthle cell carcinoma), small interfering RNA knockdown of RTN4IP1 was used to determine its role in regulating the hallmarks of malignant cell phenotype (cellular proliferation, migration, apoptosis, invasion, tumor spheroid formation, anchorage independent growth).
Results:
We found RTN4IP1 mRNA expression was significantly down-regulated in follicular and papillary thyroid cancer as compared with normal, hyperplastic, and benign thyroid neoplasms (P < .05). Moreover, RTN4IP1 mRNA expression was significantly lower in larger papillary thyroid cancers (P < .05). Small interfering RNA knockdown of RTN4IP1 expression increased cellular proliferation (2- to 4-fold) in all 3 of the cell lines tested and increased cellular invasion (1.5- to 3-fold) and migration (2- to 7.5-fold), colony formation (3- to 6-fold), and tumor spheroid formation (P < .05) in 2 of the 3 cell lines tested (FTC-133 and XTC1).
Conclusions:
This is the first study to characterize the expression and function of RTN4IP1 in cancer. Our results demonstrate RTN4IP1 is down-regulated in thyroid cancer and is associated with larger papillary thyroid cancer and that it regulates malignant cell phenotype. These findings, taken together, suggest that RTN4IP1 has a tumor-suppressive function and may regulate thyroid cancer progression.
The rate of thyroid cancer incidence is increasing, with the largest increase being in papillary thyroid cancer and small primary tumors (1, 2). Activating mutations in the MAPK pathway genes such as BRAF, RAS, and RET/PTC3 are commonly present as somatic mutations in papillary thyroid cancer, the most common type of thyroid cancer (3). In most studies, but not in all, the presence of the BRAF V600E mutation in papillary thyroid cancer has been reported to be associated with aggressive disease (3–5). Recently we also observed higher rates of the BRAF mutation over time (6). Additional markers of aggressive papillary thyroid cancer are needed to better stratify patients to determine the optimal treatment and follow-up strategy, given the increasing incidence of thyroid cancer, as well as to identify therapeutic target genes for those patients with advanced and metastatic thyroid cancer that is refractory to conventional therapy.
In our previous genome-wide expression analysis of papillary thyroid cancer, we found RTN4IP1 to be differentially expressed in tumor samples (7). It was down-regulated in papillary thyroid cancer in men, who often have more aggressive disease at diagnosis (7–9). RTN4IP1 is thought to be a mitochondrial protein because it was identified as a protein that interacts with Reticulon 4 (RTN4) (10). RTN4 is an inhibitor of neuronal regeneration and has been implicated in regulating cell cycle progression, apoptosis, and migration (11–13). Several genes, such as VHL, REST, MYC, and RET, with roles in neuronal regeneration or differentiation, have also been found to have important roles as tumor suppressor genes or oncogenes and as markers of cancer aggressiveness (13–22). Lastly, RTN4IP1 is located on chromosome 6q21, a chromosomal region frequently deleted in aggressive thyroid cancer and thus thought to be involved in thyroid cancer progression (23).
Given our previous study findings and the function of RTN4IP1 being unknown, we were interested in characterizing its expression and function in thyroid cancer. Thus, in this study we sought the following: 1) to characterize the expression of RTN4IP1 in human normal, hyperplastic, and benign and malignant thyroid tissue samples; 2) to determine whether there is any association between gene expression level and extent of disease; and 3) to determine its role in regulating the hallmarks of malignant cell phenotype (cellular proliferation, migration, apoptosis, invasion, tumor spheroid formation, and anchorage independent growth) in multiple thyroid cancer cell lines.
Materials and Methods
Thyroid tissue samples
Tissue samples were collected under a National Cancer Institute-approved tissue procurement protocol after written informed consent was obtained. Tissue samples were snap frozen immediately after thyroidectomy in liquid nitrogen and stored at −80°C. We used 9 normal thyroid, 13 Graves' disease, 12 hyperplastic nodule (adenomatous nodule), 22 follicular adenoma, 16 follicular thyroid cancer, and 47 papillary thyroid cancer samples in this study after confirmation of the tissue diagnosis by an endocrine pathologist and the tumor samples used for the analysis had greater than 80% tumor cells (Table 1).
Table 1.
Study Cohort Clinical Characteristics
| Features | Number (%) |
|---|---|
| Number of patients | 110 |
| Tissue samples analyzed | 119a |
| Sex (male/female) | 32/78 |
| Age (mean ± SD), y | 39.6 ± 11.8 |
| Papillary thyroid cancerb | 47 |
| Tumor size | |
| T1 | 15 |
| T2 | 16 |
| T3 | 7 |
| T4 | 9 |
| Lymph node metastasis | 17 (36) |
| Distant metastasis | 0 |
| Tumor mutation statusc | |
| BRAF V600E | 18 |
| KRAS | 1 |
| RET/PTC3 rearrangement | 3 |
| No mutation detected | 25 |
| Distant metastasis | 0 |
| Follicular thyroid cancer | 16 |
| Tumor size | |
| T1 | 1 |
| T2 | 2 |
| T3 | 9 |
| T4 | 4 |
| Lymph node metastasis | 0 |
| Distant metastasis | 3 (18) |
Total number of tissue samples analyzed is higher because 9 normal thyroid samples were obtained from the contralateral lobe of patients with a primary tumor (2 papillary thyroid cancer, 3 follicular adenoma, 4 follicular thyroid cancer).
Conventional papillary thyroid cancer by tumor node metastasis staging system.
Mutations in BRAF (exon 10) and hot spot mutations in KRAS and NRAS were analyzed by direct sequencing and rearrangements in RET/PTC1, RET/PTC3, and NTRK1 by nested PCR.
Thyroid cancer cell lines and culture conditions
Human papillary thyroid cancer (TPC-1), follicular thyroid cancer (FTC-133), and Hürthle cell cancer (XTC-1) cell lines were maintained in DMEM supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), Fungizone (250 ng/mL), TSH (10 IU/L), and insulin (10 μg/mL) in a 5% CO2 atmosphere at 37°C. Cells were routinely subcultured every 2–3 days.
RNA isolation and quantitative RT-PCR
Total RNA was extracted from frozen tissue samples and thyroid cancer cell lines, using TRIzol reagent (Invitrogen Life Technologies, Inc, Carlsbad, California) and the RNeasy minikit (QIAGEN, Valencia, California) according to the manufacturers' instructions. For quantitative real-time RT-PCR, 1 μg of total RNA was used for first-strand cDNA synthesis, using the Superscript III first-strand synthesis supermix for quantitative RT-PCR (Invitrogen). TaqMan primers and probes for RTN4IP1 (Hs00907217) and GUSB (Hs9999908) were purchased from Applied Biosystems (Foster City, California), and relative expression was determined using the ΔCt method (Applied Biosystems). All reactions were performed in triplicate.
Small interfering (si) RNA transfection and immunoblotting
The siRNA for human RTN4IP1 (s39464 and s39465) and scrambled negative controls (part number 4390843) were purchased from Applied Biosystems. TPC-1, FTC-133, and XTC-1 cells were transfected with each individual siRNA at a final concentration of 90 nmol/L, using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's instructions. Whole-cell lysate was prepared with radioimmunoprecipitation assay buffer and was used for RTN4IP1 protein detection by Western blot (rabbit anti-RTN4IP1).
Cell proliferation assay
Cell proliferation experiments were performed in 96-well plates in quadruplicates. Cells were transfected with individual siRNA in 96-well black plates at 2 × 103 cells/well and maintained in 200 μl serum-free media [DMEM/Ham's F-12 (1:1) supplemented with insulin (10 μg/mL), transferrin (5 μg/mL), somatostatin (10 ng/mL), and hydrocortisone (0.36 ng/mL)] in a humidified incubator. CyQuant proliferation assays were performed on each day after transfection, according to the manufacturer's instructions (Invitrogen). The cell densities in the 96-well black plates were determined using a 96-well fluorescence microplate reader (Molecular Devices, Sunnyvale, California) at 485 nm/538 nm.
Soft agar assay for colony formation
Three days after siRNA transfection, FTC-133 and XTC-1 cells were trypsinized, counted, and resuspended in culture media. Two-layered soft agar assays were performed in 6-well plates. The bottom layer of agar (2 mL/well) contained 0.6% agar (Difco agar noble; Becton, Dickinson and Co, Sparks, Maryland) in Ham's F-12 medium, supplemented with 10% fetal calf serum, penicillin (100 U/mL), streptomycin (100 μg/mL), and Fungizone (250 ng/mL). Thirty thousand cells were mixed with 1 mL of upper agar solution (0.35% agar in culture media). After 30 minutes, 1 mL of culture media was added into each well. The plates were cultured at 37°C in 5% CO2, and the media were changed twice a week. After 7 and 14 days of culture, cell colonies were stained with Crystal Violet and examined by microscopy. Colony counting was performed in 3 fields using ImageJ software (National Institutes of Health, Bethesda, Maryland).
Spheroid culture
Three days after siRNA transfection, FTC-133 and XTC-1 cells were trypsinized, counted, and resuspended in culture media and plated in an Ultra Low Cluster plate (Costar, Corning, New York) at a concentration of 3.5 × 104/well in 24-well plates. The plates were cultured at 37°C in 5% CO2, and the medium was changed every 2–3 days. At 1 and 2 weeks after plating, cells were photographed under a light microscope.
Invasion assay
Three days after siRNA transfection, FTC-133 and XTC-1 cells were trypsinized, counted, and resuspended in serum-free culture media. Cell invasion was assessed using the BD BioCoat Matrigel invasion chamber (BD Biosciences, Bedford, Massachusetts), according to the manufacturer's protocol. A total of 5 × 104 cells were seeded onto the inserts (8 μM pore sized polycarbonate membrane) coated with a thin layer of Matrigel basement membrane matrix (BD Biosciences) and control insert provided. The cells were incubated in serum-free media. The inserts were placed into a 24-well plate with 10% serum-containing culture medium as a chemoattractant. The plates were incubated for 24 hours at 37°C. Cells that invaded the Matrigel matrix to the lower surface of the membrane were fixed and stained with Diff Quik Stain (Dade Behring, Newark, New Jersey) and counted under a light microscope in 3 fields, using ImageJ software (National Institutes of Health). Invasion was calculated using the following formula: percentage invasion = number of cells in Matrigel insert/number of cells control insert, normalized to negative control.
Migration
Three days after siRNA transfection, FTC-133 and XTC-1 cells were trypsinized, counted, and resuspended in culture media. A total of 1 × 106 cells was plated in 6-well plates. The plates were incubated for 8 h at 37°C. Using a 200-μL pipette tip, 2 vertical and 2 horizontal scratches were created. Every 8 hours the wound closure was measured under a light microscope. Wound closure measurement was performed in 4 fields, using Image J software (National Institutes of Health).
Data analysis
ANOVA post hoc tests and parametric and nonparametric t tests were used for statistical analysis (GraphPad Prism, La Jolla, California).
Results
RTN4IP1 is down-regulated in papillary and follicular thyroid cancer and is associated with larger primary tumors in papillary thyroid cancer
Quantitative real-time (RT)-PCR was performed on 9 normal human thyroid tissues, 13 Graves', 12 hyperplastic nodules, 22 follicular adenoma, 16 follicular thyroid cancer, and 47 papillary thyroid cancer samples to assess RTN4IP1 mRNA expression. There was no significant difference in expression between normal, Graves', hyperplastic nodules, and follicular adenomas (Figure 1). However, there was decreased mRNA expression level in papillary thyroid cancer and follicular thyroid cancer as compared with normal, Graves', hyperplastic nodules, and follicular adenoma (P < .05) (Figure 1, A and B). There was also a significantly lower expression of RTN4IP1 with increasing primary tumor size in papillary thyroid cancer (Figure 1C). We observed no significant difference in RTN4IP1 expression by lymph node metastasis, the presence of extrathyroidal invasion, and mutational status (BRAF, KRAS, NRAS, RET/PTC1, RET/PTC3, NTRK1) in papillary thyroid cancer (Table 1).
Figure 1.
RTN4IP1 mRNA expression levels in normal, benign, and malignant thyroid tissue. A, RTN4IP1 mRNA expression in normal, Graves', nodular hyperplasia, and papillary thyroid cancer (PTC). B, Comparison of RTN4IP1 mRNA expression in normal, follicular adenoma (FA), and follicular thyroid cancer (FTC). C, RTN4IP1 mRNA expression in normal and papillary thyroid cancer (PTC) by tumor T status (tumor size: T1 = ≤2 cm; T2 = >2 cm but < 4 cm; T3 = ≥ 4 cm). The expression level is shown in −Δ Ct. Error bars indicate mean ± SD.
Effect of RTN4IP1 on malignant cell phenotype
To determine an effective strategy to assess the function of RTN4IP1, we evaluated RTN4IP1 expression levels in multiple thyroid cancer cell lines and found it to be expressed in FTC-133, TPC-1, and XTC-1 cell lines. The expression levels in the cell lines were all similar, with ΔCt ranging from 2 to 3 (relative to GUSB) (data not shown). Using 2 siRNAs (si4 and si5) to target RTN4IP1, we were able to knock down RTN4IP1 for up to 12 days, as confirmed by quantitative RT PCR and Western blot (Figure 2).
Figure 2.
RTN4IP1 expression with and without siRNA knockdown in thyroid cancer cell lines. A, Relative knockdown of RTN4IP1 mRNA expression by quantitative RT PCR. B, Representative Western blot for RTN4IP1 protein expression after knockdown (NC/C). C, Negative control siRNA; Si4/Si-1 and Si5/Si-2, siRNA targeting RTN4IP1. Percentage of RTN4IP1 mRNA and protein expression with Si4 and Si5 knockdown relative to negative control siRNA was measured by quantitative RT PCR and band densitometry measurement, respectively. The anti-RTN4IP1 antibody was purchased from Sigma-Aldrich (St Louis, Missouri; Prestige rabbit anti-RTN4IP1 antibody).
Cell proliferation was assessed in FTC-133, XTC-1, and TPC-1 cell lines. In FTC-133 and XTC-1, starting at day 3, there was an increase in cell proliferation with knockdown of RTN4IP1 relative to negative control (Figure 3). Knockdown of RTN4IP1 led to a 2- to 3-fold increase in cell number by day 7 in FTC-133, XTC-1, and TPC-1 cell lines (P < .05).
Figure 3.
The effect of RTN4IP1 knockdown on cellular proliferation in FTC-133, XTC-1, and TPC-1 thyroid cancer cell lines. Error bars indicate mean ± SD. NC, negative control siRNA. Si4 and Si5: siRNA targeting RTN4IP1.
To assess the effects of RTN4IP1 on 3-dimensional tumor formation, a spheroid formation assay was used to mimic a solid tumor. In both XTC-1 and FTC-133 cell lines, which form spheroid, knockdown of RTN4IP1 led to an increase in the size of spheroids (Figure 4A). Furthermore, knockdown of RTN4IP1 led to more densely packed spheroids, whereas negative control transfected cells formed loosely packed aggregates. This in vitro finding is consistent with the in vivo data showing lower RTN4IP1 expression was associated with larger tumor size.
Figure 4.
The effect of RTN4IP1 knockdown on tumor spheroid and colony formation. A, RTN4IP1 knockdown increased the number and size of tumor spheroid in FTC-133 and XTC-1 thyroid cancer cell lines. B, RTN4IP1 knockdown increased colon number and size in FTC-133 and XTC-1 thyroid cancer cell lines. C, Quantification of colonies formed with (Si4 and Si5) and without (NC) RTN4IP1 knockdown. TPC-1 cell lines do not form tumor spheroids and colonies in soft agar. NC, negative control siRNA; Si4 and Si5, siRNA targeting RTN4IP1.
A soft agar colony formation assay was performed to assess anchorage-independent growth in the XTC-1 and FTC-133 thyroid cancer cell lines, which form colonies in soft agar, with knockdown of RTN4IP1. We found significantly increased colony numbers in both XTC-1 and FTC-133 cell lines (Figure 4B). In FTC-133, knockdown of RTN4IP1 with si4 and si5 led to a 3.2- and 2.1-fold increase in number of colonies, respectively, at 1 week, and a 5.5- and 2.4-fold increase, respectively, at week 2 when compared with negative control (P < .05). Similarly, knockdown of RTN4IP1 with si4 and si5 in XTC-1 cells led to a 6.1- and 6.7-fold increase in number of colonies, respectively, at 1 week, and a 6.3- and 7.0-fold increase, respectively, at week 2 when compared with the negative control (P < .05) (Figure 4C). The TPC-1 cell line does not form colonies in soft agar and thus was not analyzed.
For tumor cells to form metastatic foci, they must have an enhanced ability to migrate and invade the extracellular matrix. To assess the effects of RTN4IP1 knockdown on migration, a wound-healing assay was performed. Knockdown of RTN4IP1 led to enhanced migration of cells in both FTC-133 and XTC-1 cell lines but not the TPC-1 cell line (Figure 5A). In FTC-133 cells, there was an increase in wound closure as early as 8 hours with the knockdown of RTN4IP1, with complete closure of the wound before 24 hours, whereas in the negative control, 61% of the wound was closed (P < .05) (Figure 5B). Similarly, XTC-1 cells showed an increase in wound closure. The effects were observed as early as 8 hours and were continued through the 24-hour period, with 79%–90% closure in si4 and si5 knockdown, respectively, vs 59% closure in negative control cells (P < .05) (Figure 5B).
Figure 5.
The effect of RTN4IP1 knockdown on wound closure and cellular invasion. A, RTN4IP1 knockdown decreased wound closure in FTC-133 and XTC-1 thyroid cancer cell lines. B, Quantification of wound closure with and without RTN4IP1 knockdown. C, RTN4IP1 knockdown decreased cellular invasion in FTC-133 and XTC-1 thyroid cancer cell lines. D, Quantification of cells invaded with and without RTN4IP1 knockdown. NC, negative control siRNA; Si4 and Si5, siRNA targeting RTN4IP1.
To assess the effect of RTN4IP1 knockdown on invasion, we used TPC-1, FTC-133, and XTC-1 cell lines. Knockdown of RTN4IP1 led to an increase in invasion in FTC-133 and XTC-1 cell lines but not in the TPC-1 cell line (Figure 5C). In FTC-133, knockdown of RTN4IP1 with si4 and si5 led to a 1.5- and 2.8-fold increase in the number of cells, respectively, as compared with the negative control (P < .05) (Figure 5D). Similarly, knockdown of RTN4IP1 with si4 and si5 in XTC-1 cells led to a 2.0- and 3.2-fold increase in the number of cells, respectively, compared with the negative control (P < .05) (Figure 5D).
Discussion
In this study, we characterized the expression of RTN4IP1 in normal, hyperplastic, and benign and malignant thyroid tissue samples. We also evaluated the role of RTN4IP1 in regulating the cellular proliferation, migration, invasion, tumor spheroid formation, and anchorage-independent growth. We found RTN4IP1 mRNA expression was down-regulated in follicular and papillary thyroid cancer and that this lower expression was associated with larger primary tumor size in papillary thyroid cancer. Our functional studies demonstrated that RTN4IP1 knockdown was associated with an increase in cellular proliferation in all 3 cell lines tested and increased tumor spheroid formation and size, anchorage-independent growth (increased colony size and number), and cellular migration and invasion in 2 of the 3 cell lines tested.
Our previous work identified RTN4IP1 as differentially expressed in papillary thyroid cancer by sex (7). Therefore, we were interested in determining whether the RTN4IP1 expression was dysregulated in thyroid cancer as compared with benign thyroid neoplasms and normal and hyperplastic thyroid tissue. Indeed, we found that RTN4IP1 was lower in follicular and papillary thyroid cancer, as compared with normal and hyperplastic thyroid tissue and benign thyroid neoplasm. Moreover, lower RTN4IP1 expression was associated with larger papillary thyroid cancer. These findings of the loss of RTN4IP1 may contribute to cancer progression.
This study is the first to characterize the misexpression of RTN4IP1 in cancer cells. The mechanism for the down-regulated expression of RTN4IP1 in follicular and papillary thyroid cancer is unclear, but the gene is located on chromosome 6q21, a chromosomal region frequently deleted or with loss of heterozygosity in a variety of human malignancies (including thyroid cancer) and specifically in comparisons of primary and metastatic/aggressive tumors (23–29). Furthermore, on query of the Catalogue of Somatic Mutations in Cancer database for RTN4IP1 mutations, 3 of 93 cancer samples (2 colon cancer, 1 breast cancer) analyzed are reported to have mutations.
Because RTN4IP1 was down-regulated in thyroid cancer, the expression was lowest in larger primary papillary thyroid cancer, and the function of the gene is unknown, we were interested in understanding the function of RTN4IP1 in thyroid cancer cells. We found RTN4IP1 knockdown resulted in an increase in cellular proliferation in all 3 cell lines tested and an increase in cellular migration and invasion, tumor spheroid formation, and anchorage-independent growth in 2 of the 3 thyroid cancer cell lines tested. These findings, taken together, suggest that RTN4IP1 may have a tumor-suppressive effect in cancer and may be involved in thyroid cancer progression. However, it is possible that in papillary thyroid cancer, because of the lack of effect of RTN4IP1 knockdown on cellular migration and invasion in the TPC1 cell line, the primary effect of RTN4IP1 may be on the cellular growth rather than motility or invasion.
Mitochondrial genes and metabolism have an important role in cancer initiation and progression (30). Hu et al (10) identified RTN4IP1 and demonstrated its physical interaction with RTN4 in the endoplasmic reticulum. RTN4 is a potent inhibitor of fibroblast spreading and neurite extension in the central nervous system (31). RTN4 has also been shown to attenuate migration of respiratory smooth muscle cells and endothelial cells (32–34). Based on this, we propose that RTN4IP1 effect as shown in our functional studies may be related to its interaction with RTN4. Acting as a cofactor, RTN4IP1 could regulate the function of RTN4 to influence cellular invasion and migration. Consistent with this hypothesis, other proteins associated with the RTN protein family have been shown to demonstrate a tumor-suppressive quality (35). Mechanistically, it is also possible that knockdown of RTN4IP1 could lead to a change in the apoptotic pathway of tumor cells, allowing the cells to escape programmed cell death, thereby leading to the increase in proliferation observed in our experiments.
In summary, this is the first study to characterize the expression and function of RTN4IP1 in cancer. Our results demonstrate RTN4IP1 is down-regulated in thyroid cancer and is associated with larger papillary thyroid cancer and that it regulates malignant cell phenotype (proliferation, anchorage independent growth, spheroid formation, migration, and invasion). These findings, taken together, suggest that RTN4IP1 has a tumor-suppressive function and may regulate thyroid cancer progression.
Acknowledgments
This work was supported by the Intramural Research Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health.
Disclosure Summary: The authors have nothing to disclose.
Footnotes
- FTC-133
- follicular thyroid cancer
- RT
- real time
- RTN4
- Reticulon 4
- si
- small interfering
- TPC-1
- papillary thyroid cancer
- XTC-1
- Hürthle cell cancer.
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