Additional considerations in cancer cell radioresistance, integrin αvβ3 and thyroid hormones

ABSTRACT

Background

The existence of a functional relationship between a certain thyroid hormone analogue and cancer cell radioresistance has been shown by Leith and coworkers. The hormone analogue with relevance to malignant cells’ radioresistance is tetraiodothyroacetic acid (tetrac). Tetrac is the deaminated derivative of L-thyroxine (T4), the principal product of the thyroid gland. Preclinical studies demonstrated that tetrac and chemically modified tetrac (CMT), e.g. a fluorobenzyl-conjugated tetrac analogue, restores radiosensitivity in certain radioresistant tumor cells. Due to their molecular, physico-chemical, and biological properties, actions of CMT analogues are believed to be initiated at the thyroid hormone analogue receptor site on plasma membrane integrin αvβ3.

Objective

To explore possible molecular mechanisms of the potentially therapeutically beneficial effect of CMT on cancer cells’ sensitivity to radiation, we analyzed actions of CMT analogues on expression of selected sets of genes that have been previously implicated in radioresistance of malignant cells.

Discussion and conclusions

In the current study, we report that genome-wide gene expression profiling analysis of human glioblastoma (GBM) and acute myelocytic leukemia (AML) cell lines exposed in vitro to noncytotoxic doses of CMT has identified decreased expression of discrete trios of genes each of which was previously linked to cancer cells’ radioresistance. Following the CMT treatment in AML cells, expression of PARP9, PARP15 and STAT3 genes was significantly reduced, while in GBM cells, expression of PRKDC, EGFR and CCNDI was significantly decreased by the drug. Notably, a broader spectrum of genes implicated in cancer cells’ radioresistance was observed in primary patient-derived GBM cells after the CMT treatment. Extensive additional experimental and clinical studies are indicated, including analyses of individual patient tumor genomics and of an array of different tumor types to define the sub-sets of tumors manifesting radioresistance in which tetrac-based agents may be expected to enhance therapeutic effects of radiation.

KEYWORDS:

• Tetrac
• tetraiodothyroacetic acid
• thyroid hormone integrin receptor

Introduction

Leith and coworkers in 2018 pointed out that radiation was capable of activating integrin αvβ3 in tumor cells.¹ The activated state is manifested by extension of the integrin molecule that reduces the intercellular space by fostering integrin-extracellular protein interactions, which appeared to interrupt cell division.² Chemically modified tetrac (CMT) analogues are believed to be binding to the thyroid hormone analogue receptor site on plasma membrane integrin αvβ3 interfering with integrin αvβ3 signaling.³ Recent reviews of cell signaling systems involved in cancer cell radioresistance demonstrate the complexity of the mechanisms of radioresistance.^4–11 It was of interest to extend these analyses by considerations of possible mechanistic contributions to cancer cells’ radioresistance of the signaling pathways initiated from integrin αvβ3. Cessation of cell division is likely contributed to acquisition of the radioresistant state, since non-dividing cancer cells are relatively insensitive to radiation. The authors were aware that thyroid hormone analogues have a receptor on αvβ3 and they found that tetraiodothyroacetic acid (tetrac) – a derivative of the principal product of the thyroid gland, L-thyroxine (T4) – and (CMT) analogues prevented radiation-induced activation of the integrin. These compounds did not affect the basal (folded) state of the αvβ3 protein which is associated with the relatively radiosensitive state of a cell.²

Against this background, it was of interest to determine whether expression of genes involved in radioresistance of cancer cells was significantly decreased in cancer cells treated with CMT analogues, subject to control from integrin αvβ3 by thyroid hormone analogues. If so, then the possibility existed that thyroid hormone analogues are involved in control of radioresistance beyond the issue of the activation state of integrin αvβ3. Leith and colleagues reported that thyroid hormone (as T4 or tetrac or both) modulated actions of a number of cancer cells’ radioresistance-relevant signaling pathways, likely acting at the thyrointegrin receptor on αvβ3.² These included the AKT, Wnt-ß-catenin and STAT3 pathways. It is now apparent that a panel of other signaling pathways modifies the radioresistance/radiosensitivity state of cancer cells. Among others, these additional systems include Hedgehog, ATM/ATR (ataxia-telangiectasia mutated and Rad3-related), HIF-I (hypoxia-inducible factor-I) and BRCA.^4,6,7,9 We know that these signaling systems are subject to control by T4 via the integrin in normal cells and cancer cells, but we have only recently begun to understand whether such control is relevant to radioresistance.

Materials and Methods

The software-retrieved raw data of genome-wide microarray and/or RNAseq gene expression profiling experiments were processed and subjected to an unpaired t-test with the Benjamin-Hochberg False Discovery Rate (FDR) correction. A 1.5-fold expression changes filter was applied to identify differentially expressed transcripts between the control and test conditions at a p-value <0.05. During the selection of differentially expressed genes (DEGs), both nominal and FDR adjusted p-values were of considered. Analyzed and reported data are MIAME (minimum information about a microarray experiment) compliant and the raw data have been deposited in the Gene Expression Omnibus (GEO; GSE140272; GSE140449; GSE180889; GSE180895; GSE180896; GSE183170; GSE183482; GSE183772; GSE183773; GSE183776) as detailed on the Microarray Gene Expression Data Society (MGED) website (http://www.mged.org/Workgroups/MIAME/miame.html). Overall, the workflow of the microarray analyses was modeled after a previously published contribution.¹²

Results and Discussion

We analyzed the effect of chemically modified tetrac (CMT), fb-PMT (NP751)^13,14 on expression of a panel of genes whose products are known contributors to radioresistance in standard models of established human cancer cell lines of human glioblastoma (GBM) and acute myeloid leukemia (AML). Examples of the cancer cells’ radioresistance gene panels were collected by Liu et al.⁶ and reflect the functions of various signal transduction pathways, DNA damage repair, cell cycle redistribution, epithelial–mesenchymal transition (EMT) and cancer stem cell (CSC) markers. We hypothesized that if the patterns fb-PMT action are cell line-specific, then the possibility may exist that tetrac-based agents may have broadly based applications in radiosensitizing previously radioresistant tumor cells. Notably, certain of the signal transducing factors implicated in cancer cells’ radioresistance may also be involved in other cellular defense and survival mechanisms available to cancer cells, such as anti-apoptosis or pro-angiogenesis, which are known to be affected by various CMT analogues.⁶

It has been observed that in AML cells, the action of fb-PM (NP751) caused a statistically significant (defined at the 1.5 log-fold expression changes; p < 0.05) reduction in expression of PARP15 (poly[ADP-ribose]polymerase3), PARP9 and STAT3 (signal transduction and activator of transcription 3) genes. In U87 GBM cells, drug action caused statistically significant reductions in expression of PRKDC (protein kinase, DNA-activated, catalytic polypeptide), EGFR (epidermal growth factor receptor) and CCND1 (cyclin D1) genes. This provided a genomic basis for the previously reported¹⁵ radiosensitization of U87MG brain tumor cells tetrac. Extension of these analyses to the model systems including another CMT analogue, P-bi-TAT,¹⁶ revealed that expression of certain cancer cells’ radioresistance genes is down-regulated in established human pancreatic cancer cell-line SUIT2 and in primary GBM patients-derived cancer cells (Table 1). We note that compared to established human cancer cell lines, in primary patient-derived GBM cells the CMT treatment decreased expression of a broader spectrum of genes implicated in cancer cells’ radioresistance (Table 1).

Table 1. Examples of human cancer radioresistance genes expression of which is down-regulated in various human cancer cells by synthetic chemically modified tetrac (CMT) analogues.

Gene symbol	Description	Fold expression changes (log2)	P value	CMT analogue	Cancer type	Model system
AKT1	v-akt murine thymoma viral oncogene homolog 1	−1.73	0.0308	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
AKT2	v-akt murine thymoma viral oncogene homolog 2	−1.77	0.0265	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
CCND1	cyclin D1	−0.31	0.00297	fb-PMT	Glioblastoma Multiforme	U87 established cancer cell line
CDK4	cyclin-dependent kinase 4	−1.95	0.0111	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
CDKN1A	cyclin-dependent kinase inhibitor 1A (p21, Cip1)	−2.4	0.0021	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
EGFR	epidermal growth factor receptor	−1.28	4.19E–05	fb-PMT	Glioblastoma Multiforme	U87 established cancer cell line
H2AFX	H2A histone family, member X	−4.16	4.54E–05	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
MYCN	v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog	−2.09	0.0065	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
PAK4	p21 protein (Cdc42/Rac)-activated kinase 4	−2.04	0.0073	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
PARP15	poly(ADP-ribose) polymerase family member 15	−1.88	0.0166	fb-PMT	Acute Myeloid Leukemia	K562 established cancer cell line
PARP9	poly(ADP-ribose) polymerase family member 9	−1.96	0.0088	fb-PMT	Acute Myeloid Leukemia	K562 established cancer cell line
PRKDC	protein kinase, DNA-activated, catalytic polypeptide	−1.01	0.00085	fb-PMT	Glioblastoma Multiforme	U87 established cancer cell line
RAD51	RAD51 recombinase	−2.37	0.0081	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
RPA2	replication protein A2	−1.95	0.0051	P-bi-TAT	Pancreatic cancer	SUIT2 established cancer cell line
STAT3	signal transducer and activator of transcription 3	−1.81	0.0328	fb-PMT	Acute Myeloid Leukemia	K562 established cancer cell line
TGFB1	transforming growth factor beta 1	−1.87	0.0178	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
TGFB1I1	transforming growth factor beta 1 induced transcript 1	−2.11	0.0048	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
WNT2B	wingless-type MMTV integration site family, member 7B	−1.73	0.0276	P-bi-TAT	Pancreatic cancer	SUIT2 established cancer cell line
WNT5B	wingless-type MMTV integration site family, member 5B	−1.69	0.0453	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells
WNT7B	wingless-type MMTV integration site family, member 5B	−2.13	0.0023	P-bi-TAT	Pancreatic cancer	SUIT2 established cancer cell line
XRCC3	X-ray repair complementing defective repair in Chinese hamster cells 3	−2.85	0.0021	P-bi-TAT	Glioblastoma Multiforme	Patient-derived primary cells

What may be concluded from these initial results is that CMT analogues can downregulate expression of different radioresistance-linked genes in cancer cells derived from different types of human malignancies. Normal (nonmalignant) cells contain relatively small amounts of integrin αvβ3 and the protein appears not to be activated,³ so that exposure to tetrac may be less likely to affect the viability of such cells in the radiation field. As noted in the Introduction, physiological concentrations of T4 can protect cancer cells from radiation. One possible mechanism of this phenomenon is that the principal product of the thyroid gland, T4, activates the integrin signaling; this leads to suppression of (or interference with) biological processes and molecular functions that are essential to successful radiation therapy. Consequently, tetrac-based drugs may act to block this action of T4 at the thyrointegrin and restore cancer cells’ radiosensitivity.^2,15,17

Results of gene expression profiling experiments reported in Table 1 underscore the complexity of mechanisms of radioresistance in cancer cells affected by the CMT analogues. Thus, genomic control at the gene expression level of these diverse mechanisms may exist at the thyroid hormone receptor site on integrin αvβ3, which could be affected (activated and/or repressed) nongenomically.

Abbreviations

PARP, poly(ADP-ribose)polymerase; STAT3, signal transduction and activation of transcription 3; PRKD, protein kinase, DNA activated, catalytic polypeptide; CCNDI, cyclin D protein 1, AML, acute myeloid leukemia; CMT, chemically modified tetrac; CSC, cancer stem cell; EGFR, epidermal growth factor receptor; EMT, epithelial–mesenchymal transition; GBM, glioblastoma (multiforme). Acronyms in Table 1 are defined in the Table.

Acknowledgments

The authors appreciate the contributions of Jessica Wrobel to the preparation of the manuscript.

Disclosure Statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

The author(s) reported there is no funding associated with the work featured in this article.