Thyroid and androgen receptor signaling are antagonized by CRYM in prostate cancer

Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3’-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth. Highlights Thyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by μ- Crystallin (CRYM). [18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels. Reduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients.


Introduction
PCa is the most frequently diagnosed cancer in men in the Western world. The course of PCa is largely driven by androgen receptor (AR) and, accordingly, androgen ablation therapy is a cornerstone of current therapies (1,2). A large proportion of patients with advanced PCa become resistant to ADT resulting in lethal castration resistant prostate cancer (CRPC). CRPC is mainly associated with genetic amplifications, mutations, or other aberration in the AR (3,4). Besides androgen, the role thyroid hormone thyroxine (T4) and its more active form 3,5,3'-triiodo-L-thyronine (T3) in the progression of PCa has not been comprehensively elucidated.
The thyroid hormones released into circulation are mostly bound to plasma proteins and subsequently transported to the cytosol by thyroid hormone (TH) transporters which have diverse binding affinities such as Transthyretin (TTR) and thyroxinebinding globulin (TBG) (5). The activity of TH within the cell is regulated by (i) cell uptake involving transporters such as MCT8, (ii) metabolization by DIO1 and DIO3, two members of the iodothyronine deiodinase family and (iii) sequestration via binding to other proteins (6) such as the cytoplasmic protein CRYM, which is known to bind to T3 with high-affinity (7). Intracellular thyroid hormone function in the prostate is dependent on CRYM expression levels (8,9). Previous reports demonstrated that CRYM expression was downregulated in PCa patients who underwent ADT (10) and that expression of CRYM is also downregulated in a PCa xenograft tumor model (11). It was shown that CRYM expression is particularly low in therapy refractory PCa patient biopsies (11). We corroborated this finding and showed that CRYM is responsive to androgens in the MDA PCa 2b cell line (10).
The objective of this study was to define the role of thyroid hormone and its regulation by CRYM in PCa. We hypothesized that TH signaling may play an auxiliary role in PCa progression. Here we show that CRYM expression levels are

Low µ-crystallin (CRYM) expression is a negative prognostic factor and a hallmark of PCa
We assessed CRYM expression levels in malignant and adjacent normal biopsies derived from a large PCa patient cohort by immunohistochemistry (IHC). Decreased protein expression of CRYM was observed in PCa patient samples (n=178) compared to normal prostate glands (n=178), and a further reduction in CRYM expression was observed in metastatic samples (n=17, Figure 1A). Representative IHC images of cytosolic CRYM in normal prostate, PCa and metastases are shown in Figure 1A. These findings were validated through a second independent cohort covering PCa and benign prostate tissue samples (Tuebingen Cohort, Figure 1B).
PCa specimens (n=122) had lower CRYM protein levels as compared to benign samples (n=30). Strikingly, low mRNA expression of CRYM in metastatic PCa samples compared to primary PCa tissue was observed in multiple larger PCa patient cohorts ( Figure 1C, E, F, G, H). Furthermore, CRYM expression was significantly higher in non-neoplastic prostate glands compared to primary PCa and to samples from metastasized PCa ( Figure 1C ) (12). Reduced CRYM expression also correlated with advanced Gleason scores or progressive 'N' stages of regional lymph node infiltration (supplementary Figure 1A, B, C). Together, these data indicate that loss of CRYM expression in PCa is an indicator for PCa aggressiveness. We analyzed the effect of CRYM expression on biochemical recurrence (BCR)-free survival by Kaplan-Meier analysis in 179 PCa Vienna cohort patients. Low CRYM expression was associated with a reduced time to BCR, indicating that low CRYM expression predicts poor prognosis ( Figure 1D). A single patient was also assessed for CRYM and TRβ expression longitudinally within a 4month follow-up using transurethral resections of PCa. During this period, AR and PSA expression were reduced whereas Ki-67 staining was significantly increased, suggesting progression to a highly aggressive androgen-independent neoplasia.
Indeed, we found that CRYM expression was markedly reduced and TRß level increased during progression to androgen-independency in line with our previous 5 findings (supplementary Figure 1D). Taken together, the expression and prognostic value of CRYM reveals its role in regulation of AR and TH signaling in PCa.

CRYM enables intracellular accumulation of T3 in PCa cells
To determine the effect of CRYM on intracellular accumulation of T3, we transfected CRYM negative PCa cell lines with empty vector or CRYM expression vector ( Figure   2A). Growth medium was supplemented with 10 nM T3 or vehicle, and after 48 hours, T3 levels were measured in the supernatants using a immunechemiluminescence assay. We observed significantly reduced levels of T3 in the medium of all CRYM re-expressing cells (PC3, 44%; DU-145, 22%; 22RV-1, 20% and LAPC-4, 18% Figure 2A and B) compared to controls. To confirm that this reflected uptake into cells, radioactively labeled thyroid hormone [ 125 I] T3 was supplemented to the culture media. More T3 was found in PC3 and LNCaP cell lines with CRYM overexpression compared to empty vector controls ( Figure 2C). It can be hypothesized that CRYM binds free T3 in the tumor cells, which results in increased T3 uptake and concomitantly reduced T3 levels in the growth medium.

CRYM reduces the invasive potential of PCa cells by interfering with T3
signaling WB analysis of CRYM and TRβ expression levels demonstrated that these are mutually exclusive in the androgen-independent PC-3 and DU-145 cell lines. In the androgen-dependent cell lines, both CRYM and TRβ expression could be detected ( Figure 3A). Re-introduction of CRYM in PC-3 and DU145 cells reduced TRβ expression ( Figure 3B). Next, we monitored the effect of high CRYM expression on invasive potential using matrigel-coated invasion chambers. CRYM overexpression significantly reduced invasion by 50% (p=0.0167) and 67% (p=0.0014) for PC3 and DU-145 cells, respectively ( Figure 3C). This is in line with our patient data exhibiting that CRYM protein expression is downregulated in metastasizing tumors whereas Interestingly, genes involved in AR signaling including dihydrotestosterone (DHT; n=61) and AR regulated (AR; n=46) genes were significantly downregulated ( Figure   4A). We then compared our RNA-Seq dataset of CRYM overexpressing cells to a published RNA-Seq. dataset (17) wherein the same cell line had been treated with DHT ( Figure 4B and C). Interestingly, among the overlapping, deregulated genes, 70.1% were counter-regulated, suggesting antagonistic functions for CRYM and AR signaling. In this dataset, 843 out of 3427 significantly deregulated genes overlapped with the CRYM deregulated genes ( Figure 4C). We performed IPA analysis on the DHT deregulated genes (>2-fold, q<0.05; n=145). As expected, progesterone, DHT, AR and metribolone regulated genes were strongly enriched and the respective pathways activated, whereas Bicalutamide and LY294002 associated genes were downregulated ( Figure 4C). Complementary to this approach, shRNA-mediated knock-down of CRYM in LNCaP cells resulted in a significant increase in PSA levels (38±2ng/ml as compared to 10±2 ng/ml of control, Figure 4D). Since AR protein or mRNA levels were not altered, the strong effect of CRYM overexpression on target gene expression, suggests a crosstalk between androgen and thyroid hormone signaling in PCa ( Figure 4E).

CRYM alters the choline metabolism and metabolic shift caused by T3.
Recent studies have revealed that tumor suppressor pathways such as PTEN, p53 and RB are involved in regulation of glucose and glycine metabolism resulting in  Figure   6B). A follow up analysis was performed in 87 patients that correlated with FMC uptake to biochemical recurrence and/or synchronous metastatic disease. Mean follow up time in this cohort was 508 days. Patients with biochemical recurrence or already initial metastases had significantly higher FMC uptake ( Figure 6C). ROC analysis shows the confidence interval curve of 0.77 with p<0.0001 ( Figure 6D).
These data indicate that FMC signal in prostate is indicative of BCR, and that this correlates with TRβ and CRYM expression. Our results suggest that choline is closely associated with intracellular thyroid hormone levels and that the 18 F-FMC PET/MRI tracer for the activity of thyroid hormone could be used to predict high and low risks in PCa patients. Whether FMC-PET imaging can be used as a marker for the activity of thyroid hormone metabolism in PCa needs to be tested in further studies. In summary we conclude that absence of CRYM results in an increased choline metabolism in PCa, which is a poor prognostic indicator that can noninvasively be measured in vivo by 18 F-FMC PET/MRI.

Discussion
We investigated the crosstalk of CRYM with T3 and AR signalling in PCa. We PET imaging using metabolic tracers offers the opportunity not only for non-invasive diagnostics but also long-term surveillance. Interestingly, we found that overexpression of T3-buffering CRYM leads to a reduction in intracellular choline levels in PC3 cells. CHKA has a crucial role in phospholipid metabolism through catalyzing the choline phosphorylation to form phosphocholine, a phospholipid component of bilayers in cell membranes (25). Total choline expression has been shown to be elevated in PCa tissue as compared to healthy, age matched controls (26)(27)(28). This feature has been successfully used in combination with PET/MRI to enhance the accuracy of non-invasive diagnostics (29 (7). Hypothyroidism was shown to slow down growth of aggressive breast cancer (30). Earlier reports indicated also that hypothyroidism induced by PTU in rats inhibited metastatic growth, formed smaller tumors and prolonged survival in liver cancer (31). The question of how hypothyroidism diminishes growth of PCa could be possibly addressed in the future with more specific drugs with less side effects blocking the thyroid hormone pathway that could be used in the combination with AR blockers (32,33).
In conclusion, we provide strong evidence for the protective effect of T3-buffering by CRYM in PCa. Therefore, CRYM might represent a novel biomarker for detection of good prognostic PCa. In addition, we provide a rational for how thyroid hormone metabolism could influence choline levels in PCa, which is important not only for diagnostic imaging techniques. Based on the fact that thyroid hormone signaling might act as a new oncogenic factor, our study also contributes to the understanding of aggressive PCa. Importantly new treatment avenues using novel and specific antithyroid drugs may open up and inclusion of such drugs in currently used regimens for metastatic PCa will be of interest.  The colonies were counted and photographed (Nikon D90).

Quantification of PSA and T3
Lentivirally transduced LNCaP cells were seeded at a density of 10 5  Research.

RNA and qPCR
Total RNA was isolated from cultured cells homogenised in QIAzol Lysis Reagent Statistical analysis was performed in GraphPad Prism 6.

Wound healing and invasion assay
Cells were grown in 24-well plates with cell culture inserts for wound healing assays.
After 48 hours, medium and inserts were removed to create a wound/gap. New

Cell proliferation
LNCaP and PC3 cells were seeded into 6-well plates in triplicate at a density of

NMR-Based Metabolomics Analysis of Culture Media and Cell Pellets
The culture media from PC3 were filtrated using 3-kDa cutoff Nanosep centrifugal  Figure S4B. For the cell pellet extract, first the spectral regions with VIP -CI ≥ 0.5 were determined and identified (as described above). For those metabolites whose NMR signals appear in a different spectral region, the VIP (CI) of the spectral region with the highest VIP -CI and minimum spectral overlap with other metabolites was chosen for further presentation as relative concentration ( Figure S4B).

FMC-PET/MRI patient study
In the scope of a prospective clinical phase III trial (