Skip to main content
Download PDF

Abnormal phosphorylation of human LRH1 at Ser510 predicts poor prognosis and promotes cell viability in lung squamous cell carcinoma

BMC Cancer volume 25, Article number: 764 (2025) Cite this article

Abstract

The nuclear receptor liver receptor homolog 1 (LRH1)/NR5A2 is aberrantly expressed in diverse cancer types, including liver and lung cancers. Since we previously showed that excessive phosphorylation of human LRH1 at S510 (hLRH1pS510-high) is predictable of hepatocellular carcinoma recurrence, we here clarified the clinicopathological and biological significance of hLRH1pS510-high in lung cancer. By immunohistochemistry using an anti-hLRH1pS510 monoclonal antibody, we evaluated the hLRH1pS510 signals in 151 and 150 cases of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tissues, respectively, and performed clinicopathological analysis. hLRH1pS510 was localized in the nucleus of tumor cells in LUAD and LUSC tissues with different intensity and proportions among the patients. Of note, the strong hLRH1pS510 signal was occasionally detectable in LUAD and LUSC cells at the expanding tumor edges. A semi-quantitative analysis revealed that 28 (18.4%) and 36 (24.0%) of LUAD and LUSC cases, respectively, exhibited hLRH1pS510-high. Kaplan-Meier plots showed significant differences in the disease-free survival (DFS) between the hLRH1pS510-high and hLRH1pS510-low groups in LUSC, but not in LUAD patients. hLRH1pS510-high was also significantly correlated with recurrence in LUSC patients. Additionally, by multivariate analysis, hLRH1pS510-high represented an independent biomarker for the DFS of LUSC patients. Furthermore, the impact of hLRH1pS510 on the viability of LUSC cells was evaluated by comparing phenotypes among two distinct LUSC cell lines expressing wild-type LRH1, LRH1S510A, and LRH1S510E. Consequently, we demonstrated that phosphorylation of hLRH1S510 accelerates the viability of LUSC cells. Thus, hLRH1pS510 is attractive not only as the predictive biomarker for LUSC but also as the potential therapeutic target.

Peer Review reports

Background

Lung cancer is the second most common malignancy and the leading cause of cancer-related deaths worldwide [1, 2]. It was estimated that every year, globally, nearly 2.5 million people are diagnosed with lung cancer, and over 1.8 million patients die of this tumor. Approximately 85% of lung cancer corresponds to non-small cell lung cancer (NSCLC), of which lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) represent the first and second major subtypes, respectively [3,4,5,6]. A wide variety of genes and signaling pathways are known to be altered in both LUAD and LUSC [7,8,9,10,11]. However, in sharp contrast to LUAD, the common driver mutations are rarely defined in LUSC, resulting in few targeted therapies for this histological subtype [12,13,14]. Additionally, immunotherapy is effective for a subset of LUSC patients, but not for the majority of them [10, 15]. It is therefore of great importance to identify predictive biomarkers and therapeutic targets for LUSC is of vital importance.

The nuclear receptor superfamily transcriptionally regulates the expression of numerous target genes [16, 17]. The activity of nuclear receptors is coordinated not only by ligand binding but also by post-translational modifications such as phosphorylation [18,19,20,21]. We previously uncovered the AKT-phosphorylation motif (RXXS) in retinoic acid receptors (RARs) and estrogen receptors (ERs), and found that phosphorylation of mouse RARγS379 and human ERαS518 regulates the activities of these receptors independently of their ligands [22, 23]. We also showed that ERαS518 is responsible for controlling the ERα activity in breast and endometrial cancer cells, as well as for driving endometrial cancer progression [22, 24]. Moreover, our findings indicated that phosphorylation of human liver X receptor β (LXRβ) at S432 contributes to accelerating breast cancer metabolism and progression [25]. Of note, these phosphorylation motifs are conserved in 14 of 48 members of human nuclear receptors [22], including liver receptor homolog 1 (LRH1/NR5A2), which further suggests the biological relevance of these phosphorylation sites.

The LRH1 is an orphan nuclear receptor and plays a key role in maintaining cholesterol homeostasis, steroidogenesis, reproduction, embryonic development, cell proliferation, and differentiation [26,27,28,29]. LRH1 also influences the pathogenesis of various types of malignant tumors, such as lung, liver, pancreatic, gastric, colon, breast, and prostate cancers [30, 31]. Concerning lung cancer, it is reported that high LRH1 expression is associated with poor overall and disease-free survival (DFS) in patients with NSCLC [32]. In addition, by the use of small interfering RNA, it is revealed that LRH1 stimulates self-renewal and tumorigenesis of mouse Lewis lung cancer [33]. Regarding the phosphorylation of hLRH1, S238 and S243 in the hinge region are known to be phosphorylated by extracellular signal-regulated kinases [34]. S469 in isoform 2 of hLRH1, which corresponds to S510 in isoform 1 of hLRH1, can be also phosphorylated by protein kinase A [35]. However, the absence of the phosphorylation-specific antibodies (Abs) hinders the evaluation of the relevance of these phosphorylation sites. Along this line, we have recently developed a specific monoclonal antibody (mAb) that specifically recognizes the phosphorylated form of hLRH1S510 (hLRH1pS510), and shown that hLRH1pS510-high is an independent parameter for hepatocellular carcinoma recurrence [36]. Taken together, we hypothesized that an aberrant hLRH1S510 phosphorylation participates in NSCLC advancement and could be a therapeutic target.

In the present study, we determined, by immunohistochemistry using the anti-hLRH1pS510 mAb, whether the high hLRH1pS510 is a novel biomarker to predict prognosis for LUSC and/or LUAD patients. We also verified whether hLRH1pS510 contributes to elevating cell viability in LUSC, similar to the findings showing that hERαpS518 and hLXRβpS432 promote endometrial and breast cancer progression. Moreover, we discussed the use of hLRH1pS510 as a potential therapeutic target for LUSC.

Materials and methods

Antibodies

The rat anti-hLRH1pS510 mAb was established previously [36] and used for immunohistochemistry. Mouse anti-hLRH1 mAb (H2325, Perseus Proteomics, Tokyo, Japan), HRP-conjugated anti-FLAG mouse mAb (A8592, MERCK, Rahway, NJ, USA), and HRP-conjugated anti-actin beta mouse mAb (sc-47778 HRP, Santa Cruz Biotechnology, Dallas, TX, USA) were used for immunoblotting.

Tissue collection

Formalin-fixed paraffin-embedded (FFPE) tissues were obtained from 301 patients with LUAD and LUSC who had undergone a lung resection between Jan 2012 and Dec 2017 at Fukushima Medical University Hospital or Takeda General Hospital. The clinicopathological characteristics of the patients were determined through a combination of histopathological diagnosis of the surgical specimens and diagnostic imaging including chest contrast CT, whole-body PET scan, and head contrast MRI, in accordance with the eighth edition of the Union for International Cancer Control (UICC)/American Joint Committee on Cancer (AJCC) TNM classification for lung cancer (Supplementary Table S1). The study was approved by the Research Ethics Committee of Fukushima Medical University (approval code, 2020-058; approval date, Mar 16, 2021).

Immunohistochemistry and analysis

Immunohistochemistry was performed as previously described [36]. Briefly, the deparaffinized and rehydrated FFPE tissue sections were immersed in 0.3% hydrogen peroxide in methanol for 20 min at room temperature (RT), followed by incubation in boiling citric acid buffer (pH 6.0) in a microwave. After cooling, the sections were blocked with 1% bovine serum albumin (BSA) for 30 min. They were then incubated overnight at 4℃ with the anti-hLRH1pS510 mAb [36]. The VECTASTAIN Elite ABC HRP Kit for rat (PK-6104; Vector Laboratories, Burlingame, CA, USA) was used for 3′,3′-diaminobenzidine (DAB; 347–00904, DOJINDO, Kumamoto, Japan) staining.

Immunostaining results were interpreted by two independent pathologists and one respiratory surgeon, who were blinded to clinical data, using the Allred scoring system [37]. The intensity score (IS) of the nuclei of the tumor cells was classified into four categories, ranging from 0 to 3. Subsequently, the percentage of tumor cells exhibiting staining intensity of 1 or more was calculated as a proportion score (PS), in which scores of 5, 4, 3, 2, and 1 were assigned to categories representing 67% or more, 34–66%, 10–33%, 1–10%, and less than 1%, respectively. The sum of the IS and PS was calculated to obtain a total score ranging from 0 to 8. Based on this analysis, the samples were divided into two groups: LRH1pS510-low (score ≤ 7) and LRH1pS510-high (score = 8).

Cell culture, expression vectors, and transfection

Human LUSC cell lines LK-2 (JCRB0829) and RERF-LC-AI (RCB0444) cells were obtained from JCRB cell bank (Osaka, Japan) and RIKEN BioResource Research Center (Saitama, Japan), respectively. LK-2 cells were grown in Dulbecco’s modified Eagle medium (DMEM; 043-30085, FUJIFILM Wako Pure Chemical, Osaka, Japan) supplemented with 10% fetal bovine serum (FBS; MERCK) and a 1% penicillin-streptomycin mixture (168-23191; FUJIFILM Wako Pure Chemical), while RERF-LC-AI cells were maintained in RPMI 1640 medium (189–02025, FUJIFILM Wako Pure Chemical).

The overexpression cell lines were generated by lentiviral transfection using expression vectors pCSII-hLRH1-IRES2-Venus, pCSII-hLRH1S510A-IRES2-Venus, and pCSII-hLRH1S510E-IRES2-Venus [36]. The plasmids were constructed by inserting protein-coding sequence of hLRH1 into the BamHI/NotI site of the pCSII-EF-MCS-IRES2-Venus plasmid (RDB04384, RIKEN, Wako, Japan) [36]. The two mutants were generated by a standard site-directed mutagenesis protocol. The primer pair of 5’-cgggccatcGCAatgcaggctgaagaat-3’ and 5’- cagcctgcatTGCgatggcccggatttc-3’ was used for hLRH1S510A, while 5’- cgggccatcGAAatgcaggctgaagaat-3’ and 5’- tcagcctgcatTTCgatggcccggatttc-3’ were selected for hLRH1S510E.

Lentiviral vectors were produced by transfecting 1.0 × 107 cells of 293T cells with 10 µg of the CSII plasmids containing the target genes, 5 µg of psPAX2 (#12260, Addgene, Watertown, MA, USA), and 5 µg of pCMV-VSV-G (#8454, Addgene) using Polyethylenimine Max (PEI Max; 24765-1, Cosmo Bio, Tokyo, Japan). Culture media containing recombinant lentiviruses were harvested 72 h after transfection and exposed directly to LK-2 and RERF-LC-AI cells. After seven more days and three times-passages, the cells were used for further analysis.

Immunoblot

Immunoblot was performed as previously described [25]. In brief, whole cell lysates were collected using CellLytic MT Cell Lysis Reagent (C3228, MERCK), followed by sonication with three or four bursts of 5–10 s. They were denatured by SDS (sodium dodecyl sulfate–poly-acrylamide gel electrophoresis)-containing buffer and incubated for 10 min at 95ºC. These samples were resolved by SDS-PAGE, and electrophoretically transferred onto a polyvinylidene difluoride (PVDF) membrane. The membranes were saturated with PVDF Blocking Reagent for Can Get Signal (NYPBR01, TOYOBO) for 30 min, and rinsed with Tris-buffered saline (TBS) containing 0.1% Tween 20. They were then incubated with a primary antibody solution diluted in Can Get Signal Solution 1 (NKB-101, TOYOBO, Osaka, Japan) overnight at 4ºC, and subjected to one-hour incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies diluted in Can Get Signal Solution 2 (NKB-101, TOYOBO). After rinsing again, they were exposed to EzWestLumi One (WSE-7110, ATTO, Tokyo, Japan). Each membrane was exposed to 10% H2O2 for inactivating HRP, and rehybridized with anti-beta actin antibody as loading controls.

Cell viability assay

The total viable cell count was quantified by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) Kit (G3582, Promega, Madison, WI, USA). Two thousand cells were seeded on 96-well plates, and the reagent was added to each well after 48 h, followed by measurement of the absorbance at 490 nm.

Statistical analyses

We used the chi-squared test to evaluate the relationship between LRH1pS510-high and various clinicopathological parameters. Survival analysis was performed using the Kaplan-Meier method, and differences between the groups were analyzed using the log-rank test. The Cox regression multivariate model was used to detect the independent predictors of survival. Statistical significance for cell viability was analyzed by Welch’s t-test. Two-tailed P-values < 0.05 were considered to indicate a statistically significant result. All statistical analyses were performed using SPSS version 26.0 software (IBM, Armonk, NY, USA).

Results

The hLRH1pS510 signals in LUAD, LUSC, and non-tumor lung tissues

We first examined the hLRH1pS510 signals in 301 specimens of human NSCLC tissues (LUAD and LUSC subjects) by immunohistochemistry using the specific anti-hLRH1pS510 mAb [36]. hLRH1pS510 appeared to be observed in the nuclei of LUAD and LUSC cells, but the intensity and proportion varied among the subjects (Fig. 1A and B; Supplementary Figure S1A, B, E, and F). Upon the semi-quantification using the Allred score, 28 of 151 LUAD cases (18.4%) showed LRH1pS510-high, while the remaining 124 cases (81.6%) possessed LRH1pS510-low (Supplementary Figure S1C, D). On the other hand, among 150 LUSC subjects, 36 and 114 cases (24.0% and 76.0%) revealed high and low hLRH1pS510, respectively (Supplementary Figure S1G, H).

Fig. 1
figure 1

Representative immunohistological images of hLRH1pS510 in LUAD and LUSC tissues. The LUAD (A) and LUSC (B) tissues were immunohistochemically stained with the anti-hLRH1pS510 mAb. HE, hematoxylin-eosin. Scale bars, 100 μm

Full size image

Interestingly, the robust hLRH1pS510 signal was occasionally detectable in LUAD and LUSC cells at the expanding tumor edges, including the invasive fronts (Fig. 2A). It is also noted that the weak or moderate hLRH1pS510 signal was observed in parenchymal (e.g., bronchial epithelial cells) and non-parenchymal cells (e.g., lymphocytes) in close proximity to LUAD and LUSC tissues exhibiting the high hLRH1pS510 signal, in good agreement with our previous observations in both non-neoplastic liver tissues adjacent to the hLRH1pS510-high hepatocellular carcinoma [36]. Conversely, no hLRH1pS510 signal was detected in the surrounding non-tumor cells distant from the hLRH1pS510-high LUAD and LUSC tissues (Fig. 2B).

Fig. 2
figure 2

The strong hLRH1pS510 signals in the expanding fronts of LUAD and LUSC nests and the weak to moderate ones in the nearby non-tumor lung tissues. The LUAD and LUSC tissues were immunohistochemically stained with the anti-hLRH1pS510 mAb. (A) The tumor regions, including their invasive fronts, are indicated. Green arrowheads show positive nuclear signals in non-tumor cells close to LUAD and LUSC tissues possessing the strong hLRH1pS510 signal. (B) Representative hLRH1pS510 images in non-neoplastic lung tissues distant from tumor areas. HE, hematoxylin-eosin. Scale bars, 200 μm (A) and 100 μm (B)

Full size image

hLRH1pS510-high is associated with poor prognosis in patients with LUSC

Kaplan-Meier plots exhibited significant differences in the disease-free survival (DFS) between the LRH1pS510-high and LRH1pS510-low groups of LUSC (Fig. 3A). The 10-year DFS rates in the LRH1pS510-high and LRH1pS510-low groups were 54.5% and 76.5%, respectively. In addition, the overall survival (OS) values of the LRH1pS510-high LUSC subjects were prominently lower than those of the LRH1pS510-low subjects, though the differences were not significant (Fig. 3B). By contrast, there were no significant differences in DFS and OS between the LRH1pS510-high and LRH1pS510-low groups of LUAD patients (Supplementary Figure S2A, B).

Fig. 3
figure 3

hLRH1pS510-high is associated with poor prognosis in LUSC patients. The disease-free survival (A) and overall survival (B) for hLRH1pS510-high and hLRH1pS510-low in LUSC subjects are indicated

Full size image

Among the clinicopathological factors, LRH1pS510-high significantly correlated to extent of resection (P = 0.004) and recurrence (P = 0.020) in LUSC subjects, but not to older age, gender, tumor size (> 3 cm), pT2/3/4, pathological grade G2/G3, vascular invasion, lymphatic invasion, pleural invasion, lymph node metastasis, pathological stage II/III, EGFR (epidermal growth factor receptor) gene mutation, PD-L1 (programmed cell death-ligand 1) expression or distant metastasis (Table 1). Alternatively, LRH1pS510-high was associated with tumor size (P = 0.017) and vascular invasion (P = 0.030) in LUAD patients but not with older age, gender, smoking history, extent of resection, pT2/3/4, pathological grade G2/G3, lymphatic invasion, pleural invasion, lymph node metastasis, pathological stage II/III, postoperative adjuvant therapy, EGFR gene mutation, PD-L1 expression, recurrence, distant metastasis or molecular target therapy (Supplementary Table S2).

Table 1 Relationship between the hLRH1pS510 signal and clinicopathological factors in patients with LUSC (n = 150)
Full size table

hLRH1pS510-high represents an independent prognostic factor of LUSC

Upon univariate analysis, lobectomy (hazard ratio [HR] = 0.469, 95% confidence interval [CI] 0.229–0.961, P = 0.039), lymphatic invasion (HR = 2.191, 95% CI 1.170–4.103, P = 0.014), pleural invasion (HR = 3.362, 95% CI 1.850–6.264, P < 0.001), lymph node metastasis (HR = 2.516, 95% CI 1.298–4.878, P = 0.006), and LRH1pS510-high (HR = 1.970, 95% CI 1.038–3.737, P = 0.038) were significant prognostic factors for the DFS of LUSC patients (Table 2). On the other hand, older age, gender, tumor size, pT2/3/4, pathological grade G2/G3, vascular invasion, or pathological stage II/III were not prognostic markers for the DFS of LUSC subjects.

Table 2 Univariate and multivariate analyses of recurrence-free survival in LUSC
Full size table

Cox multivariate analysis revealed that lobectomy (HR = 0.194, 95% CI 0.007–0.485, P < 0.001), pleural invasion (HR = 3.582, 95% CI 1.578–8.130, P = 0.002), and LRH1pS510-high (HR = 2.668, 95% CI 1.337–5.325, P = 0.005) were independent prognostic parameters for the DFS of LUSC patients (Table 2).

LRH1pS510 increases cell viability in LUSC

To gain insight into the biological relevance of LRH1pS510 in LUSC cells, we generated LK-2 and RERF-LC-AI cells expressing EGFP, wild-type LRH1, LRH1S510A (an S510 phosphorylation-deficient LRH1 mutant), and LRH1S510E (an S510 phosphorylation-mimetic LRH1 mutant). In the latter two, LRH1S510 was substituted for an alanine or glutamic acid residue (Fig. 4A). It should be noteworthy that the level of LRH1 protein was similar among LK-2:LRH1, LK-2:LRH1S510A and LK-2:LRH1S510E cells, as well as among RERF-LC-AI:LRH1, RERF-LC-AI:LRH1S510A and RERF-LC-AI:LRH1S510E cells (Fig. 4B). Overexpression of these wild-type and mutant LRH1 did not influence their morphological appearances (Fig. 4C). Importantly, the viability in LK-2:LRH1S510A and LK-2:LRH1S510E cells was significantly decreased and increased, respectively, compared with that in LK-2:LRH1 cells (Fig. 4D). Similarly, the relative levels of the viability in RERF-LC-AI:LRH1S510A and RERF-LC-AI:LRH1S510E cells were significantly reduced and elevated, respectively, compared with that in RERF-LC-AI:LRH1 cells.

Fig. 4
figure 4

hLRH1pS510 is critical for cell viability in LUSC cells. (A) The construct of hLRH1 expression vector. EF-1α, elongation factor-1α; Fl, Flag; IRES, internal ribosome entry site. (B) Western blot analysis for the indicated proteins in the revealed LK-2 and BERF cells. (C) Representative phase contrast and GFP images in the indicated cell lines. Scale bar, 100 μm. (D) The relative levels of cell viability in the indicated cells are shown in the histograms (mean ± SD, n = 8)

Full size image

Discussion

We previously developed the mAb that selectively recognizes hLRH1pS510[36]. The outstanding specificity of this mAb was demonstrated by the following results: (1) it bound to the pS510-LRH1 polypeptide in a dose-dependent manner by ELISA (enzyme-linked immunosorbent assay) but not to the non-phosphorylated one; (2) by immunohistochemistry, it detected nuclear signals in hLRH1-expressing 293T cells but not in hLRH1S510A-expressing ones; (3) its positive immunoreactivity disappeared upon antibody absorption; (4) In both ELISA and immunohistochemistry, its response to pS510-LRH1 was completely prevented by lambda protein phosphatase, and the inhibition was recovered upon addition of phosphatase inhibitor [36]. By immunohistochemical analysis using this anti-hLRH1pS510 mAb, we in the present work showed that the nuclear hLRH1pS510 signal is observed in LUAD and LUSC tissues, with varied intensity score and proportion score. Upon semiquantification, the high hLRH1pS510 signal was detected in 28/152 LUAD cases (18.4%) and 36/150 LUSC subjects (24.0%). Intriguingly, the strong hLRH1pS510 signal was detectable in LUAD and LUSC cells of the expanding tumor periphery, including the invasive fronts, implying that hLRH1pS510 may be involved in LUAD and LUSC invasion. It is also noteworthy that the faint or intermediate hLRH1pS510 signal was observed in both parenchymal and non-parenchymal healthy cells adjacent to the hLRH1pS510-high LUAD and LUSC tissues. However, such signal was not observed in normal cells apart from those in the cancer nests. Taken together with analogous observations in hepatocellular carcinoma [36], these results reinforce that cancer tissues may secret some factors capable of phosphorylating hLRH1 at S510 in the surrounding non-tumor cells.

Another conclusion in this study is that aberrant phosphorylation of hLRH1S510 is able to predict poor outcomes in LUSC patients (Fig. 5A). The evidence for this conclusion was based on the following results: (1) the 10-year DFS rate in the hLRH1pS510-high group of the LUSC patients was 54.5% and significantly low compared with that in the hLRH1pS510-low group; (2) LRH1pS510-high was significantly associated with recurrence in LUSC subjects; (3) upon univariate analysis, high hLRH1pS510 exhibited a significant prognostic factor for the DFS of LUSC patients; (4) multivariate analysis revealed that hLRH1pS510-high was an independent prognostic marker for the DFS of LUSC subjects. On the other hand, hLRH1pS510-high in LUAD significantly correlated to tumor size and vascular invasion, yet it was not associated with poor prognosis. The reason for these differences between LUSC and LUAD is unknown, but it may be explained by the variation in patient characteristics between two NSCLC subtypes. Large-scale and/or prospective clinical analyses would be required to obtain more solid conclusions on the clinicopathological relevance of hLRH1pS510-high in patients with LUSC and LUAD.

Fig. 5
figure 5

The clinicopathological and biological relevance of hLRH1pS510 is in LUSC. (A) A flowchart indicating that high hLRH1pS510 is predictable of poor prognosis in patients with LUSC. (B) A Schema showing that AKT-dependent phosphorylation of hLRH1S510 contributes to LUSC progression

Full size image

Importantly, we have uncovered that hLRH1pS510 promotes the viability in LUSC cells (Fig. 5B). This conclusion was drawn by comparing phenotypes among two distinct LUSC cell lines expressing EGFP, wild-type LRH1, LRH1S510A, and LRH1S510E. The mechanisms by which hLRH1pS510 affects on the LUSC viability and the patient’s prognosis are currently unknown. However, with regard to the significance of the conserved AKT-phosphorylation motif (RXXS) in nuclear receptors, we previously showed that phosphorylation of mRARγS379 results in the dissociation of the nuclear receptor corepressor (NCoR) from RAR response elements in the promoter of target genes, thereby activating their expression and inducing epithelial differentiation [22]. We also reported that an aberrant phosphorylation of hERα at S518 is essential for the control of target gene expression in breast and endometrial cancer cells, as well as for endometrial cancer progression, such as cell proliferation and migration [22, 24]. Furthermore, we demonstrated that phosphorylation of hLXRβ at S432 is definitely involved in promoting breast cancer metabolism and progression [25]. Thus, similar to these three nuclear receptors, phosphorylation of hLRH1S510 may regulate the transcriptional activity, leading to driving LUSC progression. Further in vitro/vivo studies are required to clarify the underlying molecular basis of the effects of hLRH1pS510.

Our clinicopathological and biological analyses indicated that hLRH1pS510 could be a therapeutic target for LUSC (Fig. 5B). Since there are few common driver mutations and targeted therapies for LUSC [12,13,14], it would be highly beneficial to verify whether hLRH1pS510 is a drug target for LUSC. Moreover, given that AKT is activated in many types of cancers, including LUSC [10, 15, 38, 39], it is reasonable to focus on the AKT-consensus phosphorylation site hLRH1S510 as a potential therapeutic target for LUSC. To this end, our anti-hLRH1pS510 mAb, along with LK-2:LRH1, LK-2:LRH1S510A, RERF-LC-AI:LRH1, and RERF-LC-AI:LRH1S510A cells, would provide valuable tools for drug screening. Additional studies are necessary to develop drugs targeting hLRH1pS510 in different types of cancers, including LUSC.

In conclusion, the present study demonstrated that aberrant hLRH1S510 phosphorylation is predictable of poor prognosis and promotes cell viability in LUSC. Further analyses are required to determine whether S510 phosphorylation of hLRH1 accelerates malignant phenotypes other than promoting cell viability in LUSC, as well as the use of hLRH1pS510 as a potential therapeutic target for LUSC.

Data availability

All data generated or analyzed during this study are included in this article and its online supplementary material. Further enquiries can be directed to the corresponding authors.

Abbreviations

Abs:

Antibodies

BSA:

Bovine serum albumin

CDR:

Complementarity-determining region

CI:

Confidence interval

DFS:

Disease-free survival

ELISA:

Enzyme-linked immunosorbent assay

ERs:

Estrogen receptors

FBS:

Fetal bovine serum

FFPE:

Formalin-fixed paraffin-embedded

HR:

Hazard ratio

HRP:

Horseradish peroxidase

LXRβ:

Liver X receptorβ

LRH1:

Liver receptor homolog 1

LUAD:

Lung adenocarcinoma

LUSC:

Lung squamous cell carcinoma

mAb:

Monoclonal antibody

NCoR:

Nuclear receptor corepressor

NR5A2:

Nuclear receptor 5A2

NSCLC:

Non-small cell lung cancer

OS:

Overall survival

RARs:

Retinoic acid receptors

RFS:

Recurrence-free survival

TBS:

Tris-buffered saline

References

  1. Bray F, Laversanne M, Sung H, Ferlay J, Siegel RL, Soerjomataram I, et al. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2024;74:229–63.

    Article  PubMed  Google Scholar 

  2. Grigorean VT, Cristian DA. Cancer-Yesterday, today, tomorrow. Medicina. 2022;59.

  3. Molina JR, Yang P, Cassivi SD, Schild SE, Adjei AA. Non-small cell lung cancer: epidemiology, risk factors, treatment, and survivorship. Mayo Clin Proc. 2008;83:584–94.

    Article  PubMed  Google Scholar 

  4. Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG. Introduction to the 2015 world health organization classification of tumors of the lung, pleura, thymus, and heart. J Thorac Oncology: Official Publication Int Association Study Lung Cancer. 2015;10:1240–2.

    Article  Google Scholar 

  5. Thai AA, Solomon BJ, Sequist LV, Gainor JF, Heist RS. Lung cancer. Lancet. 2021;398:535–54.

    Article  PubMed  Google Scholar 

  6. Zhang Y, Vaccarella S, Morgan E, Li M, Etxeberria J, Chokunonga E, et al. Global variations in lung cancer incidence by histological subtype in 2020: a population-based study. Lancet Oncol. 2023;24:1206–18.

    Article  PubMed  Google Scholar 

  7. Herbst RS, Morgensztern D, Boshoff C. The biology and management of non-small cell lung cancer. Nature. 2018;553:446–54.

    Article  CAS  PubMed  Google Scholar 

  8. Pan Y, Han H, Labbe KE, Zhang H, Wong K-K. Recent advances in preclinical models for lung squamous cell carcinoma. Oncogene. 2021;40:2817–29.

    Article  CAS  PubMed  Google Scholar 

  9. Wang M, Herbst RS, Boshoff C. Toward personalized treatment approaches for non-small-cell lung cancer. Nat Med. 2021;27:1345–56.

    Article  CAS  PubMed  Google Scholar 

  10. Lau SCM, Pan Y, Velcheti V, Wong KK. Squamous cell lung cancer: current landscape and future therapeutic options. Cancer Cell. 2022;40:1279–93.

    Article  CAS  PubMed  Google Scholar 

  11. Hendriks LEL, Remon J, Faivre-Finn C, Garassino MC, Heymach JV, Kerr KM, et al. Non-small-cell lung cancer. Nat Rev Dis Primers. 2024;10:71.

    Article  PubMed  Google Scholar 

  12. Cancer Genome Atlas Research Network. Comprehensive genomic characterization of squamous cell lung cancers. Nature. 2012;489:519–25.

    Article  Google Scholar 

  13. Kim Y, Hammerman PS, Kim J, Yoon J-A, Lee Y, Sun J-M, et al. Integrative and comparative genomic analysis of lung squamous cell carcinomas in East Asian patients. J Clin Oncol. 2014;32:121–8.

    Article  CAS  PubMed  Google Scholar 

  14. Campbell JD, Alexandrov A, Kim J, Wala J, Berger AH, Pedamallu CS, et al. Distinct patterns of somatic genome alterations in lung adenocarcinomas and squamous cell carcinomas. Nat Genet. 2016;48:607–16.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Pan D, Hu AY, Antonia SJ, Li C-Y. A gene mutation signature predicting immunotherapy benefits in patients with NSCLC. J Thorac Oncol. 2021;16:419–27.

    Article  CAS  PubMed  Google Scholar 

  16. Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schütz G, Umesono K, et al. The nuclear receptor superfamily: the second decade. Cell. 1995;83:835–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Evans RM, Mangelsdorf DJ. Nuclear receptors, RXR, and the big Bang. Cell. 2014;157:255–66.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Kato S, Endoh H, Masuhiro Y, Kitamoto T, Uchiyama S, Sasaki H, et al. Activation of the Estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science. 1995;270:1491–4.

    Article  CAS  PubMed  Google Scholar 

  19. Hu E, Kim JB, Sarraf P, Spiegelman BM. Inhibition of adipogenesis through MAP kinase-mediated phosphorylation of PPARgamma. Science. 1996;274:2100–3.

    Article  CAS  PubMed  Google Scholar 

  20. Treviño LS, Weigel NL. Phosphorylation: a fundamental regulator of steroid receptor action. Trends Endocrinol Metab. 2013;24:515–24.

    Article  PubMed  PubMed Central  Google Scholar 

  21. Filtz TM, Vogel WK, Leid M. Regulation of transcription factor activity by interconnected post-translational modifications. Trends Pharmacol Sci. 2014;35:76–85.

    Article  CAS  PubMed  Google Scholar 

  22. Sugimoto K, Ichikawa-Tomikawa N, Kashiwagi K, Endo C, Tanaka S, Sawada N, et al. Cell adhesion signals regulate the nuclear receptor activity. Proc Natl Acad Sci U S A. 2019;116:24600–9.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sugimoto K, Chiba H. The claudin-transcription factor signaling pathway. Tissue Barriers. 2021;9:1908109.

    Article  PubMed  PubMed Central  Google Scholar 

  24. Kojima M, Sugimoto K, Kobayashi M, Ichikawa-Tomikawa N, Kashiwagi K, Watanabe T, et al. Aberrant Claudin-6-Adhesion signaling promotes endometrial Cancer progression via Estrogen receptor α. Mol Cancer Res. 2021;19:1208–20.

    Article  CAS  PubMed  Google Scholar 

  25. Murakami-Nishimagi Y, Sugimoto K, Kobayashi M, Tachibana K, Kojima M, Okano M, et al. Claudin-4-adhesion signaling drives breast cancer metabolism and progression via liver X receptor Î’. Breast Cancer Res. 2023;25:41.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Ikeda Y, Lala DS, Luo X, Kim E, Moisan MP, Parker KL. Characterization of the mouse FTZ-F1 gene, which encodes a key regulator of steroid hydroxylase gene expression. Mol Endocrinol. 1993;7:852–60.

    CAS  PubMed  Google Scholar 

  27. Fayard E, Auwerx J, Schoonjans K. LRH-1: an orphan nuclear receptor involved in development, metabolism and steroidogenesis. Trends Cell Biol. 2004;14:250–60.

    Article  CAS  PubMed  Google Scholar 

  28. Zhang C, Large MJ, Duggavathi R, DeMayo FJ, Lydon JP, Schoonjans K, et al. Liver receptor homolog-1 is essential for pregnancy. Nat Med. 2013;19:1061–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Stein S, Schoonjans K. Molecular basis for the regulation of the nuclear receptor LRH-1. Curr Opin Cell Biol. 2015;33:26–34.

    Article  CAS  PubMed  Google Scholar 

  30. Nadolny C, Dong X. Liver receptor homolog-1 (LRH-1): a potential therapeutic target for cancer. Cancer Biol Ther. 2015;16:997–1004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Meinsohn M-C, Smith OE, Bertolin K, Murphy BD. The orphan nuclear receptors steroidogenic Factor-1 and liver receptor Homolog-1: structure, regulation, and essential roles in mammalian reproduction. Physiol Rev. 2019;99:1249–79.

    Article  CAS  PubMed  Google Scholar 

  32. Liu Y, Xing Y, Wang H, Yan S, Wang X, Cai L. LRH1 as a promising prognostic biomarker and predictor of metastasis in patients with non-small cell lung cancer. Thorac Cancer. 2018;9:1725–32.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ye T, Li J, Sun Z, Liu Y, Kong L, Zhou S, et al. Nr5a2 promotes cancer stem cell properties and tumorigenesis in nonsmall cell lung cancer by regulating Nanog. Cancer Med. 2019;8:1232–45.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lee Y-K, Choi Y-H, Chua S, Park YJ, Moore DD. Phosphorylation of the hinge domain of the nuclear hormone receptor LRH-1 stimulates transactivation. J Biol Chem. 2006;281:7850–5.

  35. Bouchard MF, Taniguchi H, Viger RS. Protein kinase A-dependent synergism between GATA factors and the nuclear receptor, liver receptor homolog-1, regulates human aromatase (CYP19) PII promoter activity in breast cancer cells. Endocrinology. 2005;146:4905–16

  36. Nishimagi A, Kobayashi M, Sugimoto K, Kofunato Y, Sato N, Haga J, et al. Aberrant phosphorylation of human LRH1 at Serine 510 is predictable of hepatocellular carcinoma recurrence. Clin Exp Med. 2023;23:4985–95.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Mod Pathol. 1998;11:155–68.

    CAS  PubMed  Google Scholar 

  38. Dotto GP, Rustgi AK. Squamous cell cancers: A unified perspective on biology and genetics. Cancer Cell. 2016;29:622–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Song M, Bode AM, Dong Z, Lee M-H. AKT as a therapeutic target for Cancer. Cancer Res. 2019;79:1019–31.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

The authors thank Mr. Joji Kai, Ms. Seiko Watanabe, and Ms. Keiko Watari, Fukushima Medical University, for their technical assistance, as well as the Scientific English Editing Section of Fukushima Medical University (Fukushima, Japan) for their help with the manuscript. We also appreciate Drs. Takumi Yamaura and Yoshiko Yamaguchi, Takeda General Hospital, for preparing specimens.

Funding

This work was supported by JSPS KAKENHI (grant numbers 23H02703 and 22K08896) and the Takeda Science Foundation.

Author information

Author notes

  1. Hyato Mine and Kotaro Sugimoto contributed equally to this work.

Authors and Affiliations

  1. Department of Basic Pathology, Fukushima Medical University School of Medicine, Fukushima, 960- 1295, Japan

    Hayato Mine, Kotaro Sugimoto, Makoto Kobayashi & Hideki Chiba

  2. Department of Chest Surgery, Fukushima Medical University School of Medicine, Fukushima, 960- 1295, Japan

    Hayato Mine, Hironori Takagi, Naoyuki Okabe, Satoshi Muto & Hiroyuki Suzuki

  3. Department of Diagnostic Pathology, Fukushima Medical University School of Medicine, Fukushima, 960-1295, Japan

    Yasuyuki Kobayashi & Yuko Hashimoto

Authors
  1. Hayato Mine
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Kotaro Sugimoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Makoto Kobayashi
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Hironori Takagi
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Naoyuki Okabe
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Satoshi Muto
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Yasuyuki Kobayashi
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Yuko Hashimoto
    View author publications

    You can also search for this author inPubMed Google Scholar

  9. Hiroyuki Suzuki
    View author publications

    You can also search for this author inPubMed Google Scholar

  10. Hideki Chiba
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

HM: Investigation, Visualization, Validation. KS: Funding acquisition, Investigation, Visualization, Supervision, Validation, Writing–Original Draft, Writing – Review & Editing. MK: Investigation, Validation. HT: Validation, Resources. NO: Validation, Resources. YM: Validation, Resources. YS: Validation, Resources. YK: Validation. YH: Validation. HS: Validation, Resources. HC: Conceptualization, Funding acquisition, Project administration, Supervision, Validation, Writing–Original Draft, Writing – Review & Editing.

Corresponding authors

Correspondence to Kotaro Sugimoto or Hideki Chiba.

Ethics declarations

Ethics approval

This study was approved by the Research Ethics Committee of Fukushima Medical University (approval code, 2020-058; approval date, Mar 16, 2021). The research was conducted in accordance with the 1964 Helsinki Declaration or comparable standards. Informed consent (broad consent) was obtained from all of the participants in this study. Since it was conducted as a retrospective study using cases with a follow-up period of more than five years, the patients had already died or stopped visiting the hospital. The experimental protocol has been disclosed on the website, and the patients or their representatives were able to decline to participate in the survey if they wanted.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1

Supplementary Material 2

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mine, H., Sugimoto, K., Kobayashi, M. et al. Abnormal phosphorylation of human LRH1 at Ser510 predicts poor prognosis and promotes cell viability in lung squamous cell carcinoma. BMC Cancer 25, 764 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14160-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12885-025-14160-6

Keywords

BMC Cancer

ISSN: 1471-2407

Contact us