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. Author manuscript; available in PMC: 2022 Oct 1.
Published in final edited form as: Oncogene. 2022 Mar 7;41(16):2315–2325. doi: 10.1038/s41388-022-02242-9

Regulation of cancer stem cell activity by thyroid hormone receptor β

Woo Kyung Lee Doolittle 1, Xuguang Zhu 1, Sunmi Park 1, Yuelin Jack Zhu 2, Li Zhao 1, Paul Meltzer, Sheue-yann Cheng 1,✉
PMCID: PMC9018601  NIHMSID: NIHMS1788863  PMID: 35256781

Abstract

Increasing numbers of cancer stem cell markers have been recently identified. It is not known, however, whether a member of the nuclear receptor superfamily, thyroid hormone receptor β (TRβ), can function to regulate cancer stem cell (CSC) activity. Using anaplastic thyroid cancer cells (ATC) as a model, we highlight the role of TRβ in CSC activity. ATC is one of the most aggressive solid cancers in humans and is resistant to currently available therapeutics. Recent studies provide evidence that CSC activity underlies aggressiveness and therapeutic resistance of ATC. Here we show that TRβ inhibits CSC activity by suppressing tumor-sphere formation of human ATC cells and their tumor-initiating capacity. TRβ suppresses the expression of CSC regulators, including ALDH, KLF2, SOX2, b-catenin, and ABCG2, in ATC cell-induced xenograft tumors. Single-cell transcriptomic analysis shows that TRβ reduces CSC population in ATC-induced xenograft tumors. Analysis of The Cancer Genome Atlas (TCGA) database demonstrates that the inhibition of CSC capacity by TRβ contributes to favorable clinical outcomes in human cancer. Our studies show that TRβ is a newly identified transcription regulator that acts to suppress CSC activity and that TRβ could be considered as a molecular target for therapeutic intervention of ATC.

INTRODUCTION

Accumulated lines of evidence support the existence of cancer stem cells (CSCs) in many human cancer types, including leukemia [1], breast cancer [2], colorectal cancer [3] and brain tumors [4]. CSCs are a subpopulation of cancer cells with self-renewal capabilities and multilineage differentiation that can fuel cancer progression with high tumorigenic potency [5]. CSCs can adapt easily to changes in their microenvironment and are more resistant to conventional therapies than other cells within a tumor. CSC resistance can be induced secondary to radio- and chemotherapy or even after chemotherapy cessation.

Anaplastic thyroid cancer (ATC) is one of the most aggressive solid cancers in humans. Because ATC is extremely fast-growing, most cases present as stage IV disease, making the majority of patients with ATC ineligible for surgery [6]. ATC is usually resistant to conventional radioactive iodine therapy or chemotherapy, and there are currently no established therapeutic options to improve overall survival of these patients [7, 8]. The median survival of ATC patients is only three to five months after diagnosis [9, 10]. As of 2021, several new tyrosine kinase inhibitors (TKIs) are under evaluation in phase II clinical trials. Only a combination of dabrafenib and trametinib is approved by the U.S. Food and Drug Administration for patients with BRAF-mutated ATC. However, results from a phase II clinical trial show no definite evidence of benefits to survival [11]. Recent studies provide evidence to support the notion that perpetual self-renewing, re-initiating, and evolving tumor development driven by CSCs underlies the aggressiveness and treatment resistance of ATC [12]. These findings provided an opportunity to use ATC as model to elucidate the regulatory role of thyroid hormone receptor β (TRβ) in CSC activity.

TRβ is a transcription factor, critically important in growth, development, and differentiation. Numerous reports demonstrate that reduced expression of TRβ and its mutations are frequently observed in human cancers, including hepatocellular carcinoma, renal cell carcinoma, breast cancer, and pituitary tumors [13–16]. Studies show that mice expressing C-terminal TRβ mutations spontaneously develop thyroid cancer [17], indicating that loss of normal functions of TRβ results in it acting as an oncogene in thyroid cancer. Moreover, silencing of the THRB gene expression by hypermethylation of its promotor region [18–21] or by microRNA regulatory mechanisms [22] was found in diverse human cancers, suggesting TRβ can function as a tumor suppressor. In these studies, however, it has not been fully elucidated whether TRβ can act to regulate CSC activity in human cancer.

In this present study, we explored whether TRβ can regulate CSC activity using ATC cells as a model. We uncovered that the THRB expression is low in thyroid tumors that have high expression of the stemness-related genes. Consistent with the previous studies where THRB gene expression is silenced or reduced in cancers [13–16], we found that two ATC cell lines, THJ-11T and −16T, established from human ATC patients, had barely detectable TRβ proteins, but they expressed high levels of CSC regulators. When TRβ was exogenously expressed in ATC cells, the expression of numerous stem cell markers was inhibited, and the formation of tumor-spheres was reduced. The initiation capacity of xenograft tumors by THJ-11T and −16T was strongly suppressed by TRβ. Single-cell RNA-seq analysis of xenograft tumors showed that CSC numbers were reduced by TRβ. The findings that the expression of critical CSC regulators, such as aldehyde dehydrogenases (ALDH) are suppressed by thyroid hormone, T3, supports TRβ as a newly identified transcription factor that can suppress the CSC activity. These findings raise the possibility that targeting TRβ and its downstream signaling could be explored for potentially effective treatment of ATC.

RESULTS

Human ATC cells exhibit high activity of cancer stem cells

To assess the presence of CSC in the two human ATC cells, THJ-11T and −16T, which could contribute to the aggressiveness of ATC, we used the tumor-sphere formation assay. The assay has been extensively used by the investigators to assess CSC activity [23]. On day 5, while no spheres were detected from normal thyroid 96N cell line (Fig. 1A-I, top panel a), tumor-spheres were clearly visible for 11T and 16T cells, respectively (top panels b and c). On day 10, while again no spheres were found in 96N cells, increased size of 11T-tumor-spheres was found (lower panels, b). Tumor-spheres of 16T were also clearly visible on day 10, with larger size than those found in 11T cells (lower panels c). The size differences in tumor-spheres were apparent in the quantitative comparison shown in Fig. 1A-II. On day 5, the size of tumor-spheres of 16T cells was larger than 11T cells (A-II-a). The size differences of tumor-spheres between the two cell lines were larger on day 10 (A-II-b) than on day 5, reflecting stronger CSC activity in 16T than 11T cells.

Fig. 1. ATC exhibits high stemness, thereby facilitating its initiation and growth.

Fig. 1

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A Tumor-sphere formation assay showing self-renewal capacity of CSCs in normal thyroid cells (96N) and ATC cells, 11T and 16T (I; n = 5, respectively), and its quantitative analyses (II). B Gross image and tumor incidence (I), growth curve (II), and weight (III) of xenograft tumors derived from monolayer-cultured cells and tumor-spheres of 11T. C Gross image and tumor incidence (I), growth curve (II), and weight (III) of xenograft tumors derived from monolayer-cultured cells and tumor-spheres of 16T. The dotted boxes in B-I and C-I indicate the biggest tumors in each group that had to be dissected a week earlier than other tumors. D Representative IHC images (I) for CSCs markers, ALDH1/2 and CD44, in monolayer-cultured cells and tumor-spheres of 11T (i) and 16T (ii), respectively, and FACS analysis showing the enrichment of ALDH1/2high (II) and CD44high (III) cells in the tumor-spheres. Red arrows indicate ALDH1/2high (f and h) or CD44high (j and l) cell clusters inside tumor-spheres. Significant differences are indicated by asterisks (P < 0.05 [*], P < 0.01 [**], P < 0.001 [***], and P < 0.0001 [****]). Data represent the mean ± SD. Black and gray scale bars represents 50 μm and 10 mm, respectively. Abbreviations: ALDH Aldehyde dehydrogenase, IHC Immunohistochemical analysis, ATC anaplastic thyroid cancer, CSCs cancer stem cells, ns not significant.

In vivo xenograft studies further validated that ATC cells exhibit CSC activity. The ability for CSC to initiate tumor development was compared between monolayer cells and isolated tumor-spheres. Nude mice inoculated with isolated 11T-tumor-spheres enriched with CSCs rapidly developed larger xenograft tumors than those inoculated with monolayer cells with only a small population of CSCs (Fig. 1B-I, -II and -III). Consistently, 16T-tumor-spheres with strong CSCs initiated larger tumors than the monolayer cells (Fig. 1C-I, -II, and -III).

The detection of CSC markers ALDH and CD44 validated that the tumor-spheres inoculated to induce xenograft tumors were enriched with CSC activity as compared with monolayer cells (Fig. 1D-I, panels f and j for 11T tumor-spheres and panels h and l for 16T tumor-spheres, respectively). High ALDH activity (Fig. 1D-II) and high CD44 expression (Fig. 1D-III), as analyzed by fluorescence-activated cell sorting (FACS) analysis, further illustrated that tumor-spheres were enriched with high CSC activity. Taken together, these findings indicate that the presence of CSCs is crucial for tumor initiation in ATC.

TRβ suppresses activity of CSCs in human ATC cells

Consistent with the observations from associated studies showing that the expression of the thyroid hormone receptors (TRs) is frequently suppressed in human cancer [16], 11T and 16T ATC cells did not express detectable TRβ (Fig. 2A-I-a, lanes 1 and 5, respectively). To study the functions of TRβ in ATC cells, we stably expressed TRβ in 11T and 16T cells. Three 11T-TRβ clones (#1, #3, and #11) and four 16T-TRβ clones (W2N, #10, #13, and #17) shown to express TRβ proteins were analyzed. High levels of stem cell markers, such as ALDH and SOX2, were detected in 11T (Fig. 2A-I-i-b and -c, lane 1) and 16T cells (Fig. 2A-I-ii-b and -c, lane 5). Strikingly, the expressed TRβ totally suppressed the expression of four subtypes of ALDHs (ALDH1A1, ALDH1A2, ALDH1A3, and ALDH2, recognized by anti-ALDH1/2 antibodies) at the protein levels (Fig. 2A-I-b, lanes 2–4, lanes 6–9) (see also, Fig. 2A-II-a). TRβ also markedly lowered SOX2 protein levels (Fig. 2A-I-c, lanes 2–4, lanes 6–9) (see also, Fig. 2A-II-b). Importantly, the expressed TRβ significantly inhibited the proliferation of 11T (Fig. 2B-i) and 16T cells (Fig. 2B-ii), indicating that TRβ could also act as a tumor suppressor in ATC, as demonstrated previously in breast cancer cells [24].

Fig. 2. TRβ attenuates capacity of CSCs.

Fig. 2

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A Effect of TRß’s stable expression on CSC markers in 11T (i) and 16T (ii) cells (I) and quantitative analysis of the results (II). B Effect of TRß on cell proliferation in 11T (i) and 16T (ii) cells. C ALDEFLUOR assay showing the difference in ALDH activity and proportion of CSC population with high ALDH activity between the ATC cells and their TRß stably expressing cell lines (I), and its quantitative analysis (II; n = 3, respectively). D Tumor-sphere formation assay showing the effect of TRß on the self-renewal activity of CSC in 11T (I) and 16T cells (III), and quantitative analysis of the results (II and IV) (n = 5, respectively). A Western blot analyses (n = 3, respectively). B MTT proliferation assay (n = 3, respectively). Black arrow indicates a reference category for statistical comparison in each graph. Significant differences are indicated by asterisks (P < 0.05 [*], P < 0.01 [**], P < 0.001 [***], and P < 0.0001 [****]). Data represent the mean ± SD. Scale bar represents 50 μm. Abbreviations: ALDH aldehyde dehydrogenase, ATC anaplastic thyroid cancer, CSCs cancer stem cells, TRß thyroid hormone receptor beta, ns not significant.

Inhibition of ALDH activity first demonstrated that TRβ could act to regulate CSC activity. As shown in Fig. 2C-I, the three clones of TRβ-expressing 11T cells (Fig. 2C-I-i) and four clones of TRβ-expressing 16T (Fig. 2C-I-ii) all had reduced ALDH activity, as analyzed by ALDEFLUOR assay. The quantitative analysis showed that TRβ reduced 80–90% of ALDH activity in 11T cells and nearly 100% of ALDH activity in 16T cells (Fig. 2C-II). The CSC regulatory function of TRβ was further demonstrated by tumor-sphere formation assays. As shown in Fig. 2D-I-b, in three clones of TRβ-expressing 11T cells, tumor-sphere formation was totally blocked on day 5 and day 10. Quantitative data showed that all sizes of tumor-spheres, ranging from 10–25 μm to >50 μm, were blocked by TRβ on day 5 (Fig. 2D-II-a) and day 10 (Fig. 2D-II-b). In 16T cells expressing TRβ, formation of smaller tumor-spheres was partially blocked on day 5 and day 10 (Fig. 2D-III). The larger sizes of tumor-spheres (>25 μm) were nearly totally blocked by TRβ on day 5 (Fig. 2D-IV-a) and day 10 (Fig. 2D-IV-b). This in vitro data shows that TRβ acts to suppress CSC activity.

We next carried out xenograft studies to ask whether TRβ can suppress CSC activity in vivo. Figure 3A-I-a shows the induction of tumors by inoculation with a series dilution of parental 11T cells in 15 nude mice. Tumors were induced at each cell number inoculated, but as expected, the size of tumors progressively decreased when fewer cells were inoculated. In contrast, with inoculation of TRβ-expressing 11T cells (#3 or #11 clone) at the highest cell number, barely detectable tumors were visible (Fig. 3A-I-b and -c). At lower cell numbers, no tumors were observed. The suppression of tumor growth by TRβ is further illustrated in the growth curves (Fig. 3A-II). Tumors induced by parental 11T cells grew rapidly, whereas no visible growth was detected in TRβ-expressing 11T cells. Comparison of tumor weight from inoculation of 5 × 106 11T cells or TRβ-expressing cells revealed that 11T-induced tumors weighed an average of 500 mg, while the “bumps” derived from TRβ-expressing 11T cells weighed only a few mg (Fig. 3A-III).

Fig. 3. TRβ blocks the tumor-initiating capacity of CSCs in ATC.

Fig. 3

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A Gross images (I), growth curves (II), weight (III), and H&E staining (IV) of 11T- and 11T-TRß-induced xenograft tumors. B Gross images (I), growth curves (II), and weight (III) of 16T- and 16T-TRß-induced xenograft tumors. C Representative IHC images for proliferation markers (I) and quantitative analyses for the IHC results (II; n = 4, respectively) in the 16T and 16T-TRß xenograft tissues. D Representative IHC images for CSCs markers (I) and quantitative analyses for the IHC results (II; n = 4, respectively) in 11T and 11T-TRß xenograft tissues. E Representative IHC images for CSC markers (I) and quantitative analyses for the IHC results (II; n = 4, respectively) in 16T and 16T-TRß xenograft tissues. Black and white arrowheads in A-IV indicate vessel formation inside the tumor. Black arrows in A-III and B-III indicate a reference category for statistical comparison. Significant differences are indicated by asterisks (P < 0.05 [*], P < 0.01 [**], P < 0.001 [***], and P < 0.0001 [****]). Data represent the mean ± SEM (A and B) or SD (C–E) Black and red scale bars represent 25 μm and 10 mm, respectively. Abbreviations: TRβ thyroid hormone receptor β, ALDH aldehyde dehydrogenase, ATC anaplastic thyroid cancer, CSCs cancer stem cells, IHC immunohistochemistry.

To assess the extent to which TRβ suppressed the tumor growth, we analyzed the histological features of tumors induced by 11T parental cells and the “bumps” dissected out from inoculation of 11T-TRβ cells. The 11T parental tumors (Fig. 3A-IV, a–d panels) exhibited typical features of ATC, such as high grade of nuclear atypia, marked cellular pleomorphism, and necrosis inside the tumor, and we also found well-developed blood vessels (Fig. 3A-IV, a and c panels, black and white arrows) inside the tumor. Notably, the 11T-TRβ “bumps” were a part of normal mouse skeletal muscle. No evidence of tumor cells in these 11T-TRβ “bumps” was detected (Fig. 3A-IV, e–h panels), indicating that TRβ completely blocked the tumor initiation capacity in the 11T cells. We further carried out similar xenograft studies in 16T cells. Again, we found that TRβ suppressed tumor growth when inoculated in a cell-density dependent manner (Fig. 3B-I, compare panels b and c with a). Tumor growth induced by 16T cells was clearly inhibited by TRβ (Fig. 3B-II), and the tumor weight was reduced by 80–90% in 16T-TRβ cells as compared with parental 16T cells (Fig. 3B-III). Further evidence that TRβ acted to inhibit tumor growth included virtually nondetectable Ki-67 in 16T-TRβ tumors (Fig. 3C-I-e, see also quantitative comparison in Fig. 3C-II-a), compared to abundantly expressed Ki-67 in 16T-induced tumors (Fig. 3C-I-b). Furthermore, cyclin D1, a cell-cycle regulator, while abundantly expressed in tumors induced by parental 16T cells (Fig. 3C-I-c), was barely detectable in 16T-TRβ cells (Fig. 3C-I-f, see also quantitative comparison in Fig. 3C-II-b). Taken together, these results clearly indicate that TRβ acts to suppress tumor cell proliferation and block tumor growth.

We next explored the possibility that TRβ can block tumor initiation by inhibiting CSC activity. To this end, we analyzed the data from an in vivo limiting dilution assay, shown in Figs. 3A-I and 3B-I, to determine the tumor-initiating capacity of CSCs in 11T- and 16T-TRβ cells, respectively. CSC frequencies were calculated as previously reported [25]. We found that the tumor-initiating capacity of 11T-TRβ cells (two clones #3 and #11) was markedly decreased (~926-fold) as compared with parental 11T cells (Table S1, left panel). The tumor-initiating capacity of 16T-TRβ cells was 123-fold and 65-fold lower in clone #10 and W2N, respectively, compared to parental 16T cells (Table S1, right panel). Additional evidence that TRβ suppressed the CSC tumor-initiating capacity was shown by the reduction of CSC markers in TRβ-expressing cells. Immunohistochemical analysis showed that ALDH1/2, SOX2, CD44 and β-catenin were highly abundant in 11T-induced tumors (Fig. 3D-I, panels b, c, d and e, respectively). In contrast, virtually no ALDH1/2, SOX2, CD44 or β-catenin was detectable in “remnant/bumps” at the site where 11T-TRβ cells were inoculated (Fig. 3D-I, panels g, h, i, and j; see also quantitative comparison, Fig. 3D-II). Similarly, we also found that in 16T-TRβ tumors, ALDH1/2, SOX2, and CD44 were barely detectable as compared with 16T parental tumors (Fig. 3E-I, panel c versus i, d versus j, e versus k; also see Fig. 3E-II, panels a, b and c). β-catenin was reduced by ~70% in tumors induced by 16T-TRβ cells as compared with that of 16T cells (Fig. 3E-I, panel f versus l and Fig. 3E-II, panel d). Taken together, these results indicate that TRβ inhibits the tumor initiation capacity of CSC.

Single-cell transcriptomic analysis demonstrates reduction of CSC population by TRβ

To demonstrate directly that TRβ suppressed the activity of CSCs, we compared the gene expression profiles of CSC markers between the tumors induced by 16T and 16T-TRβ cells (Fig. 4A). We found that CSC markers, such as ALDH1A1, ALDH1A2, ALDH1A3, KLF2, ABCG2, SOX2, and CD44 were decreased in 16T-TRβ tumors, ranging from 20% to 98% (Fig. 4B) as compared with 16T parental tumors, indicating that TRβ suppressed the expression of critical CSC markers. It is interesting to note that there was no significant difference in the expression of the CTNNB1 gene at the mRNA levels between parental 16T and 16T-TRβ tumors (Fig. 4B). However, we found that β-catenin, encoded by the CTNNB1 gene, was markedly lower in the TRβ tumors (Fig. 3E-I, panels l versus f and Fig. 3E-II). These results suggested that there was post-transcriptional and/or post-translational regulation in the expression of the CTNNB1 gene in the xenograft tumors induced by the 16T-TRβ expressing cells. The decreased β-catenin protein levels contributed to lowering the CSC activity.

Fig. 4. TRβ reduces both capacity and population size of CSCs in vivo.

Fig. 4

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A A schematic diagram showing the experimental design. B RT-qPCR showing the differences of mRNA expression of CSCs markers between 16T and 16T-TRß xenograft tumors (n = 6, respectively). C ALDEFLUOR assay showing the ALDH activities of the single tumor cells dissociated from 16T and 16T-TRß xenograft tumors (I), and quantitative analyses of the results (II). D Tumor-sphere formation assay showing the self-renewal ability of the single tumor cells dissociated from 16T and 16T-TRß xenograft tumors (I), and quantitative analyses of the results (II). E t-SNE plot of single-cell RNA sequencing and its pie chart showing the population of CSCs and other types of cells in 16T (I) and 16T-TRß (II) xenograft tumors. Black arrow indicates a reference category for statistical comparison in each graph. Significant differences are indicated by asterisks (P < 0.01 [**] and P < 0.0001 [****]). Data represent the mean ± SD. Black and red scale bars represent 50 μm and 10 mm, respectively. Abbreviations: TRβ thyroid hormone receptor β, ALDH aldehyde dehydrogenase, CSCs cancer stem cells, MSCs mesenchymal stem cells.

We characterized the single cells isolated from 16T and 16T-TRβ xenograft tumors by their self-renewal and clonal tumor initiation capacity using ALDEFLUOR and tumor-sphere formation assays [26]. The parental single 16T cells showed high ALDH activity (Fig. 4C-I and -II) and large tumor-spheres (Fig. 4D-I and -II). In contrast, the two clones of 16T-TRβ cells consistently showed markedly reduced ALDH activity (Fig. 4C-I and -II) and failed to form tumor-spheres (Fig. 4D-I and -II), indicating the lack of CSC activity.

To gain a comprehensive understanding of the regulation of CSCs by TRβ, we performed single-cell RNA sequencing (scRNA-seq) analysis using the single cells dissociated from 16T parental and 16T-TRβ tumors. We obtained single-cell transcriptomes for 2,774 cells from 16T parental tumors and for 7,271 cells from 16T-TRβ tumors. Cell types for the clusters were annotated according to an expression reference data set, the Human Primary Cell Atlas [27], using the SingleR Bioconductor package. Notably, in 16T parental tumors (Fig. 4E-I), we identified CSCs (9.85%), mesenchymal stem cells (MSCs, 39.21%), epithelial tumor cells (40.43%), and mouse skin epithelial cells (10.5%). By contrast, in 16T-TRβ tumors (Fig. 4E-II), only 3.45% of cells were CSCs, representing a 3-fold reduction of CSCs in TRβ-expressing cells. The other three clusters of cells were identified as myofibroblasts (35.47%), myeloid cells (43.46%), and fibroblasts (17.62%). The contrasting cell clusters of tumor cells and MSC cells identified in 16T-induced tumors versus cell clusters of the fibroblasts, myofibroblasts, and myeloid cells in 16T-TRβ tumors suggests the remodeling of the cell landscape toward a tumor-free milieu, reflecting the CSC-inhibiting capacity of TRβ.

TRβ suppresses the expression of ALDH genes in ATC cells

Mounting evidence suggests that ALDH not only is a marker for stem cells but also regulates cellular functions related to self-renewal, expansion, differentiation, and resistance to drugs and radiation [28, 29]. Since the expression of ALDH was markedly suppressed at the protein level in TRβ-expressing cells (Fig. 2A) and in TRβ tumors (Fig. 3D, E), this prompted us to examine whether thyroid hormone (T3) can further affect the TRβ’s regulatory activity. In the absence of T3, ALDH isotypes, −1A1, −1A2, −1A3, and −2 proteins (recognized by the antibody against ALDH1/2), were below the detectable levels in 11T-TRß as compared with parental 11T cells (Fig. 5A-i-b, lane 1 versus lanes 3 and 5). The undetectable ALDH in 11T-TRß did not allow us to observe the extent of changes in the presence of T3 (Fig. 5A-i-b). The total inhibitory effect of ALDH isotypes by TRβ, with or without T3, was also observed in 16T-TRβ cells (Fig. 5A-ii-b).

Fig. 5. T3 potentiates transcriptional inhibition of ALDH by TRβ in ATC cells.

Fig. 5

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A Effect of T3 treatment on protein expression of ALDH1/2 in 11T (i), 16T (ii) and their TRß stably expressing cell lines (n = 3, respectively). B The effect of T3-bound TRß on mRNA expression of ALDH1A1 (a), ALDH1A2 (b), ALDH1A3 (c), and ALDH2 (d) in 11T (I) and 16T (II) cells (n = 6, respectively). Significant differences are indicated by asterisks (P < 0.05 [*], P < 0.01 [**], P < 0.001 [***], and P < 0.0001 [****]). Data represent the mean ± SD. Abbreviations: TRβ thyroid hormone receptor β, ALDH aldehyde dehydrogenase, ATC anaplastic thyroid cancer, ns not significant.

We next analyzed the effect of T3 on expression of these four ALDH genes at the mRNA levels. Two regulatory patterns were discerned among these four genes. One pattern was that without T3, TRβ acted to potently suppress the expression of ALDH1A1 and ALDH1A3 (Fig. 5B-I, panel a and c; bar 2 versus 1), and T3 further augmented the inhibitory effect (bars 4 versus 2). The other pattern was that without T3, TRβ could not suppress the expression of ALDH1A2 and ALDH2 mRNA, but T3 led to 50–70% suppression of these two genes (Fig. 5B-I, panel b and d; bars 4 versus 2). Similarly, in 16T cells, TRβ suppressed the expression of ALDH1A1 and ALDH1A3, and T3 further potentiated the suppression effect (Fig. 5B-II, panel a and c; bar 4 versus 2). Figure 5B-II also shows that the expression of ALDH1A2 and ALDH2 was suppressed by T3 (bar 4 versus 2). These results show that the expression of four isoforms of ALDH genes, critical CSC regulators, are suppressed by TRβ, which is further augmented by T3. Similar T3 regulating profiles were also found for the expression of other CSC markers such as SOX2, CTNNB1, KLF2, ABCG2, and CD44 genes (Fig. S2).

Clinical relevance of CSC-suppressing role of TRβ in human cancer

Analysis of gene expression data available in the public domain [30] provided new insights into the significance of the TRβ’s CSC suppression role in human cancers. As far as we know, no comprehensive and sufficiently large database of ATC is available for us to explore the clinical relevance of CSC-suppressing role of TRβ. We turned our attention to the available large database of PTC in The Cancer Genome Atlas-Thyroid Cancer (TCGA-THCA, patients: 505, normal subjects: 59) in order to determine the association of TRβ with stemness in thyroid cancer. We ranked tumor samples according to the expression of the THRB gene and sub-categorized them into 4 groups in order. We assigned the top 25% and lowest 25% as high and low THRB expression groups, respectively. Remarkably, we found that the expression of 16 cancer stemness-related genes (ALDH1A1, KLF2, ABCG2, TCF7, LEF1, CD44, POU5F1, NANOG, MSI1, MBD3, EZH2, THY1, NES, WNT1, VEGFA, and CXCR4) was consistently higher in tumors with low THRB expression than in those with high THRB expression (Fig. 6A). This reverse association between the expression of THRB and stemness-related genes supports the suppressor role of TRβ in CSC activity.

Fig. 6. TRβ is negatively associated with cancer stemness.

Fig. 6

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A Comparisons of expression of stemness-related genes between tumors with high and low THRB expression (Transcripts per million). B Downregulation of THRB mRNA expression in thyroid cancer (a), lung adenocarcinoma (b), kidney cancer (c), and glioma (d), compared to each normal tissue. C Multiple correlation analyses showing the inverse correlation between THRB gene and CSC markers in TCGA-THCA, -BRCA, -GBMLGG, -KIPAN, and -LUAD. Pearson correlation coefficient was used for the analyses. A–C TCGA-THCA data analyses. Transcriptome data from patients with THCA (n = 505), ACC (n = 79), BRCA (n = 1092), CESC (n = 304), COAD (n = 385), GBMLGG (n = 696), HNSC (n = 522), KIPAN (KICH + KIRC + KIRP, n = 891), LIHC (n = 361), LUAD (n = 516), OV (n = 303), PAAD (n = 147), PRAD (n = 497), SARC (n = 263), SKCM (n = 462), STAD (n = 415), and UCEC (n = 545) were analyzed. Significant differences are indicated by asterisks (P < 0.05 [*], P < 0.01 [**], P < 0.001 [***], and P < 0.0001 [****]). Data represent the mean ± SD. Abbreviations: ACC adrenocortical carcinoma, BRCA breast invasive carcinoma, CSCs cancer stem cells, CESC cervical and endocervical cancers, COAD colon adenocarcinoma, GBMLGG glioma, HNSC head and neck squamous cell carcinoma, KIPAN pan-kidney cancer cohort, KICH kidney chromophobe, KIRC kidney renal clear cell carcinoma, KIRP kidney renal papillary cell carcinoma, LIHC liver hepatocellular carcinoma, LUAD lung adenocarcinoma, OV ovarian serous cystadenocarcinoma, PAAD pancreatic adenocarcinoma, PRAD prostate adenocarcinoma, SARC sarcoma, SKCM skin cutaneous melanoma, STAD stomach adenocarcinoma, THCA thyroid carcinoma, THRB thyroid hormone receptor β, UCEC uterine corpus endometrial carcinoma.

At the top of this list was the ALDH1A1 gene, which we show to be nearly totally suppressed (Figs. 2A, 2C, and 4B) by elevated expression of TRβ. The suppressed expression of KLF2 and ABCG2 by elevated TRβ was validated in tumors (Fig. 4B). We show that β-catenin was markedly suppressed by elevated TRβ in ATC tumors (Fig. 3D, E), which would lead to the suppressed expression of the downstream target genes TCF7 and LEF1. The suppression of CD44 and ABCG2 by elevated TRβ was further validated by FACS analysis (Fig. S1-A and -B, respectively). These data show that low TRβ expression is tightly linked to cancer stemness in thyroid cancer.

We further investigated whether the suppressor role of TRβ in CSC activity and its clinical implications can be extended to other cancer types. Indeed, we found that THRB mRNA expression was significantly lower in thyroid cancer (Fig. 6B-a), lung adenocarcinoma (Fig. 6B-b), kidney cancer (Fig. 6B-c), and glioma (Fig. 6B-d), as compared with respective normal tissue. Importantly, the inverse correlation between THRB and CSC markers was also evident in many types of human cancers, such as breast cancer (BRCA), glioma (GMMLGG), pan-kidney cancer cohort (KIPAN), and lung adenocarcinoma (LUAD), in addition to thyroid cancer (THCA) (Fig. 6C). These observations suggest that TRβ can also function as a suppressor of CSC in human cancers other than thyroid cancer. These findings suggest that cancer cells gain proliferative advantages by silencing the expression of the THRB gene or by mutating the THRB gene to lose its normal tumor suppressing functions.

DISCUSSION

Increasing lines of evidence presented in the past decades support the hypothesis that a small population of cells in a cancer has self-renewal and tumor initiation properties. These cells, known as CSCs, are responsible for tumor initiation, aggressive growth, clonal expansion, recurrence, and resistance to chemotherapy and radiation therapy. ATC is one of the most lethal human malignancies. Although ATC is relatively rare, with only about 2% of all thyroid cancer cases, it accounts for more than 50% of all thyroid cancer deaths every year [7, 8]. The aggressiveness and resistance to available therapeutics suggest that CSC can exist in ATC to contribute to high mortality. Indeed, using validated human ATC cell lines isolated from ATC patients, we detected one important characteristics of CSC, the formation of tumor-spheres. The CSC-enriched tumor-spheres, when injected into nude mice, rapidly initiated more aggressive tumor growth than non-CSC-enriched monolayer cells did. The CSC-enriched tumor-spheres were populated with cells exhibiting strong ALDH activity and CD44 CSC markers (Fig. 1). Earlier, CSC activity was reported to be present in ATC cells (e.g., THJ-11T, THJ-16T, THJ-21T, THJ-29T and SW1736) [31, 32]. The side population enriched with CD133 in other ATC cell lines (e.g., C643 cells), also supports that CSC exists in ATC [33]. However, the present study provided more direct evidence for the existence of CSCs in ATC by single-cell transcriptomic analysis, showing 9.85% of total cells from in vivo xenograft tumors induced by 16T cells were CSC (Fig. 4).

Earlier observations show that the loss of heterozygosity, deletion, and decreased expression of the THRB gene are associated with diverse human cancers [16, 34], suggesting that TRβ can function as a tumor suppressor. Subsequently, findings from many cell-based and in vivo studies further support this notion. Consistent with our findings, reduced expression of the THRB in human ATC cells was also reported [35, 36], further supporting the role of TRβ as a tumor suppressor. Compelling evidence originates from extensive studies of mutant mice expressing a dominant-negative C-terminal frameshift mutant of TRβ, PV (ThrbPV/PV mice), in that the loss of normal functions of TRβ drives carcinogenesis of the thyroid [17] and facilitates the aberrant growth of the breast [37] and the pituitary gland [38]. Other collaborative evidence came from the studies demonstrating that overexpressed TRβ inhibits tumorigenesis [24, 32, 39].

The present studies provided compelling evidence to demonstrate a tumor suppressor role of TRβ in regulating CSC activity. We found that TRβ acted to potently inhibit CSC activity by blocking tumor-sphere formation in vitro (Fig. 2) and by reducing CSC numbers and CSC tumor-initiating capacity in vivo to block tumor growth (Fig. 3). The inhibitory actions of CSC activity by TRβ were mediated by suppressing the expression of stem cell regulators, such as ALDH, KLF2, ABCG2, SOX2, β-catenin, and CD44. In particular, T3 potentiated the activity of TRβ in the inhibition of the expression of critical stem cell regulators, such as ALDH1A1, -A2, -A3 and ALDH2. Taken together, these findings show that TRβ can function as a transcription regulator to suppress CSC activity in ATC.

Our findings that TRβ acted to suppress CSC activity in ATC have significant clinical implications. Analysis of the data in TCGA-THCA showed that the expression of 16 cancer stemness-related genes (ALDH1A1, KLF2, ABCG2, TCF7, LEF1, CD44, POU5F1, NANOG, MSI1, MBD3, EZH2, THY1, NES, WNT1, VEGFA, and CXCR4) was consistently higher in tumors with low THRB expression. The results of association analyses lend additional support that TRβ can function to suppress CSC activity in thyroid cancer. Moreover, it is important to point out that THRB gene expression was lower in thyroid cancer, lung adenocarcinoma, kidney cancer, and glioma than in each normal tissue (Fig. 6B). The inverse relationship of THRB with cancer stemness was also found in other cancers, including breast cancer (Fig. 6C). A previous study by Lopez-Mateo et al. reported that TRβ inhibits self-renewal capacity of breast cancer stem cells [40]. Our in-depth analysis of breast cancer clinical data showed that the inhibitory role of TRβ on CSCs contributed to favorable clinical outcomes in breast cancer (Fig. S3). The presence of TRβ led to favorable clinical outcomes in breast cancer was also demonstrated in previous reports [32, 41]. Taken together, these observations suggest that TRβ can function as a suppressor of CSC in human cancers other than ATC. Thus, targeting TRβ is a novel therapeutic strategy to block cancer progression and to overcome therapy resistance and recurrence in human cancers.

MATERIALS AND METHODS

Cell culture

ATC cell lines (THJ-11T and −16T) and normal thyroid cell lines (THJ-96N) were provided by Dr. John A Copland III at the Mayo Foundation for Medical Education and Research [42]. Cell line authentication was validated by Short Tandem DNA Repeat (STR) as reported [42]. The cells were maintained as previously described [42, 43].

In vivo mouse xenograft study

All animal experiments were performed under protocols approved by the National Cancer Institute Animal Care and Use Committee. For xenograft studies, 6- to 8-week-old female athymic nude mice were used as previously described [44]. To estimate the tumor-initiating capacity, we prepared three different limiting dilutions of cell preparations (5 × 104, 5 × 105, and 5 × 106 cells) for each cell line in 200 μl 50% Matrigel basement membrane matrix and subcutaneously inoculated into the right flank of mice. We then monitored the frequency of tumor initiation and tumor growth for about eight weeks and euthanized the mice to dissect the tumors for further analyses. We estimated the difference in the frequency of the tumor initiation using a webtool for the extreme limiting dilution analysis (ELDA, http://bioinf.wehi.edu.au/software/elda/).

Tumor-sphere formation assay

ATC cells (2 × 103 or 4 × 103 cells) were plated in 96-well low attachment plates (Corning) as previously described [44]. The number and size of tumor-spheres were measured and photographed by a Zeiss light microscope after 5–14 days.

Flow cytometry analysis

THJ-11T, −16T cells, or their TRβ stably expressing cells were harvested, and the flow cytometry analysis was carried out similarly as described previously [44]. The list of antibodies used in the study is included in Table S2. To assay for ALDH activity, we used an ALDEFLUOR kit as per the manufacturer’s protocol [44]. All the stained cells were analyzed by Sony SA3800 spectral cell analyzer. The data analysis was done by FlowJo software 10.5.3.

Immunohistochemistry

Tissues from an xenografted tumor were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin. Immunohistochemistry (IHC) was performed as previously described [45]. The list of antibodies used in the study is included in the Table S2. Tissue slides were visualized under a microscope at a magnification of ×200, and the relative positive cell ratio was determined by NIH ImageJ software.

Single-cell dissociation from xenografts and single cell transcriptomic analysis

We dissociated the xenograft tumors into single cells using a tumor dissociation kit. Briefly, the tumors dissected from 16T parental or 16T-TRβ xenograft mice were cut into small pieces of 2–4 mm, and these tissues were transferred into the gentleMACS C Tube containing an enzyme mix (2.35 mL of RPMI-1640, 100 µL of Enzyme D, 50 µL of Enzyme R, and 12.5 µL of Enzyme A) followed by the tumor dissociation using a gentle MACS Dissociator. The dissociated cells were shortly centrifuged, resuspended, and applied to a cell strainer (70 µm) placed on a 15 mL tube, followed by washing the cell strainer with 10 mL of RPMI-1640. The cells were then centrifuged at 300 x g for seven minutes, resuspended with the AKC lysing buffer to remove erythrocytes, washed with phosphate buffer saline, and subjected to the tumor-sphere formation assay, ALDEFLUOR assay, and single-cell RNA sequencing (scRNA-seq). Further detailed methods and bioinformatics analyses are included in Supplementary Materials and Methods section.

Statistical analysis

Continuous variables were compared using a two-tailed Student’s t-test, and categorical data were compared using a two-tailed χ2 test or Fisher’s exact test. Pearson correlation coefficient was used for the multiple correlation analyses between the THRB gene and stemness-related genes. For diagraming of the results from these analyses, we used the RStudio 1.2 program. Two-way analysis of variance with Bonferroni’s post-hoc test was used to compare tumor growth in xenograft model according to the time and groups. Disease-free or overall survival was compared between high and low THRB groups using a Kaplan–Meier estimator. Statistical analysis was performed using SPSS version 23.0 or GraphPad Prism. Data are presented as the mean ± SD. All P values were two-sided throughout, and those less than 0.05 were considered statistically significant.

Further detailed methods are included in Supplementary Materials and Methods section.

Supplementary Material

Supplemental Information

ACKNOWLEDGEMENTS

We thank Drs. Zachary Rae, Michael Kelly, and Kimia Dadkhah for carrying out single-cell RNA sequencing at the Single Cell Analysis Facility, Cancer Research Technology Program, Frederick National Laboratory (Leidos Biomed), NCI. We also thank Joelle Mornini, NIH Library, for manuscript editing assistance.

FUNDING

This research is supported by the Intramural Research Program of the Center for Cancer Research, National Cancer Institute, National Institutes of Health.

Footnotes

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41388-022-02242-9.

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