Comprehensive immunohistochemical analysis of N6-methyladenosine (m6A) writers, erasers, and readers in endometrial cancer

doi:10.21203/rs.3.rs-1607617/v1

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Comprehensive immunohistochemical analysis of N6-methyladenosine (m6A) writers, erasers, and readers in endometrial cancer

https://doi.org/10.21203/rs.3.rs-1607617/v1

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Purpose: N6-methyladenosine (m6A) is the most frequent type of messenger RNA (mRNA) modification and is implicated in diverse physiological processes. The procedure of m6A RNA modification is regulated by a dynamic interaction of ‚writers’ (METTL3, METTL4, METTL14, WTAP, KIAA1429), ‚erasers’ (FTO, ALKBH5), and ‚readers’ (HNRNPA2B1, HNRNPC, YTHDC1, YTHDC1, YTHDF1-3). In the oncological context, alterations in m6A were identified to be critically involved in tumorigenesis, proliferation, angiogenesis, and drug resistance across diverse cancer entities including endometrial cancer (EC).

Methods: In this study, we comprehensively examined the protein expression of m6A writers, readers and erasers by immunohistochemical staining in a cohort of N=65 EC patients. Protein expression data were analyzed with regard to clinical outcomes.

Results: We identified enhanced protein expression levels of METTL3, METTL14, FTO, HNRNPA2B1, and HNRNPC, respectively to be of prognostic value and linked to a shortened overall survival in EC.

Conclusion: Overall, our study points towards dysregulated m6A modification in EC and its possibility to serve as a promising prognostic biomarker.

Endometrial Cancer

m6A

biomarker

RNA modification

Endometrial cancer (EC) represents the most common cancer of the female genital tract worldwide and accounts for more than 66,500 newly diagnosed cases and about 12,900 cancer-related deaths in the United States per year¹. According to Bokhman’s dualistic scheme, EC is historically classified into two types: endometrioid, estrogen dependent (type I) EC and non-endometrioid, non-estrogen dependent (type II) EC. Usually, type I EC displays a better prognosis than type II EC, necessitating a more extensive surgical approach and adjuvant systemic and/or radiotherapy^2–4. However, this traditional dualistic scheme has limitations to reflect EC heterogeneity regarding epidemiology, clinical course, histopathology and molecular tumorbiology^5,6. Recently, The Cancer Genome Atlas (TCGA) Network has deduced a new molecular classification based on comprehensive genomic, transcriptomic and proteomic characterization of N = 373 EC: (i) Polymerase Epsilon ultramutated (POLE), (ii) microsatellite instability hypermutated (MSI-H), (iii) copy-number low (CN low), and (iv) copy-number high (CN high)⁷. This new molecular classification provides a precise tumor-biology driven EC-subgrouping with distinct clinical courses and prognosis thereby enabling stratification of the necessary surgical approach and adjuvant therapy. However, despite recent achievements in both, tumor biological understanding and new therapeutic approaches, the overall prognosis of EC has not improved since the 1970s¹. This emphasizes the urgent need for a deeper understanding of tumorigenesis and identification of prognostic and predictive biomarkers to tailor an individualized therapy to each patient.

In this context, there is increasing evidence for posttranscriptional RNA modification to be critically involved in tumorigenesis, progression, and genesis of therapy resistance - thereby representing an interesting candidate for both, therapeutical and diagnostical applications^8–10. N6-methyladenosine (m6A) is the most abundant messenger RNA (mRNA) modification. Three different enzyme groups are involved in m6A modification: (i) Methylases (‘writers’; METTL3, METTL4, METTL14, WTAP, KIAA1429) catalyze the transfer of S-adenosyl methionine groups to RNA adenine bases. (ii) Demethylases (‘erasers’; FTO, ALKBH5) are involved in reversing the methylation process. (iii) ‘Readers’ (HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1-3) recognize m6A RNA modifications and activate downstream regulatory pathways¹¹. Research has shown altered m6A protein expression to be involved in EC ¹². The majority of data is available for the fat mass and obesity-associated protein (FTO). Increased FTO expression was demonstrated to be associated with shorter survival. Of note, FTO expression is closely related to weight gain and obesity, both representing known risk factors for EC^{13, 14}.

However, sparse is known about the remaining m6A writers, erasers, and readers and their role in EC. In this study, we aimed to comprehensively elucidate the protein expression levels of m6A writers, readers and erasers by immunohistochemistry in a cohort comprising N = 65 EC patients with regard to overall survival (OS).

Patients and specimens

The retrospective study cohort included N=65 patients with EC diagnosed at the University Hospital Bonn between 2003 and 2016, with available follow-up. Tissue was obtained from surgical specimen or endometrial sampling using a pipelle. The collection of tissue was performed within the framework of the Biobank initiative of the University Hospital Bonn. All patients provided written informed consent prior to collection of biomaterials.

Clinicopathological parameters are summarized in Table 1. Baseline characteristics were obtained from clinical routine documentation. Follow-up data were updated in April 2022. Histopathological diagnosis was established based on the World Health Organization (WHO) criteria. The 7^th TNM classification of the Union for International Cancer Control (UICC) was referred to determine the tumor stage.

The study was approved by the Ethics Committee of the University of Bonn (vote: 208/21).

Tissue microarray (TMA) construction

The TMA was generated from formalin-fixed paraffin embedded EC tissue (FFPE) specimens. To identify representative tumor areas. Sections were stained with hematoxylin and eosin (HE). Afterwards, a 1mm core biopsy (0.785mm²) was taken from the selected cancer nests for each case and arranged in TMA blocks.

Immunohistochemistry

Immunostaining of METTL3, METTL4, METTL14, WTAP, KIAA1429, FTO, ALKBH5, HNRNPA2B1, HNRNPC, YTHDC1, YTHDF1,YTHDF2, and YTHDF3 was performed on TMAs using an automated staining system (BenchMark ULTRA; Ventana Medical Systems) which performed deparaffinization, pretreatment with cell conditioning buffer (CC1 buffer, pH8), and incubation with primary antibodies (FTO (1:50; Atlas Antibodies #HPA041086), ALKBH5 (1:200; Novus #NBP1-82188), METTL3 (1:1000; Biorbyt #orb374082), METTL4 (1:40; Atlas Antibodies #HPA040061), METTL14 (1:100; Atlas Antibodies #HPA038002), WTAP (1:100; Atlas Antibodies #HPA010550), KIAA1429 (1:25; Atlas Antibodies #HPA031530), HNRNPC (1:25; Atlas Antibodies #HPA051075), HNRNPA2B1 (1:100; Atlas Antibodies #HPA001666), YTHDC1 (1:25; Atlas Antibodies #HPA036462), YTHDF1 (1:10; Biorbyt #orb179018), YTHDF2 (1:200; Biorbyt #orb39199), YTHDF3 (1:200; Biorbyt #orb374095) at 4°C overnight. Signal detection was performed with the UltraView DAB IHC Detection Kit (Ventana).

Immunohistochemical stainings were analyzed using a Olympus BX51 microscope and the Panoramic Viewer 3DHistech. Staining intensities were evaluated by two different investigators. In detail, a four-tier scoring system was applied to categorize staining intensities (0: no staining, 1: low staining, 2: moderate staining, 3: high staining). For statistical analysis, staining intensities were divided into two groups (low and high). Classification of the groups low vs. high depending on the staining intensity are provided for each antibody in Table 2.

Statistical analysis:

Kaplan-Meier survival analyses and log-rank tests were used for comparison of OS between the two groups (low vs. high expression) for each analyzed protein. Hazard ratios (HR) were estimated based on univariate Cox proportional hazards regression models for each protein and are reported together with corresponding two-sided 95% confidence intervals. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS ®) version 28 (SPSS Inc., IBM Corp.) Statistical significance was considered at a two-sided p-value of <0.05.

Immunohistochemical staining was performed in a cohort of N=65 EC patients. The mean patient’s age of the study cohort was 64.9 (+/- 11.2) years. 92.3% of the patients displayed endometrioid histology, 4.6% had serous-papillary EC, and 3.1 % showed undifferentiated histology. The median follow-up for the entire cohort was 70.7 months. Data on BMI was available for N=56 patients with a median BMI of 28.3 (+/- 8.4). A detailed clinicopathologic characterization including histopathologic grading, lymph node involvement and tumor stage according to UICC is presented in Table 1.

Immunohistochemical analyses showed a strong nuclear staining for the writers METTL3, METTL14, WTAP, and KIAA1429, the eraser FTO and also the two readers HNRNPC und HNRNPA2B1. YTHDF1-YTHDF3 and METTL4 showed a strong cytoplasmic staining. YTHDC1 displayed a cytoplasmic/membranous and also nuclear staining and ALKBH5 was stained in both, cytoplasm and nucleus.

In Kaplan-Meier survival analyses, high expression levels of METTL3, METTL14, FTO, HNRNPA2B1, and HNRNPC were significantly associated with a shorter OS (p<0.05; Figure 1). For all other analyzed proteins, Kaplan-Meier survival analyses indicated a trend towards shorter overall survival in patients with higher m6A protein expression levels. However, these effects were not statistically significant. A detailed overview of the different m6A proteins with their respective protein expression values are given in Table 2. The prognostic value of METTL3, METTL14, FTO, HNRNPA2B1, and HNRNPC was confirmed in univariate Cox regression analyses. Further correlation analyses of m6A protein expression levels with respect to possible co-factors on survival including grading and lymph node involvement did not show statistically significant values.

In summary, our results points towards dysregulated m6A expression in EC tumorigenesis. In particular, high expression levels of METTL3, MEETTL14, FTO, HNRNPA2B1, and HNRNPC were positively correlated with a shortened OS.

In the present study, we comprehensively evaluated protein expression levels of m6A writers, erasers, and readers in N = 65 EC patients. Protein expression data was analyzed with regard to clinical outcomes. We demonstrated that five m6A proteins, namely METTL3, METTL14, FTO, HNRNPA2B1 and HNRNPC, respectively, correlated significantly with a poor OS in EC. In particular, overexpression of the five respective proteins was a negative prognostic marker for survival. This effect was independent of further clinicopathological parameters including histomorphological grading or lymph node involvement.

There is increasing evidence for the crucial impact of mRNA modification in cancer development, metastatic spread, and evolution of drug resistance. The most studied RNA modification is m6A ^15,16. A recent study analyzed mRNA expression data of m6A genes in N = 548 EC samples obtained from The Cancer Genome Atlas (TCGA) database. In their analyses, the authors found significant differential expression of all assessed m6A genes in EC tissue compared to normal adjacent tissue (NAT). Moreover, higher m6A mRNA expression levels were detected in poorly differentiated compared to well differentiated tumors and linked to worse clinical outcomes¹². Consistent observations on aberrant m6A in EC were reported by another study based on m6A mRNA expression analysis¹⁷. In the aforementioned studies and further investigations, it was demonstrated that increased expression of FTO in particular was associated with poorer survival in EC^18,19. In the process of m6A RNA modification, FTO facilitates demethylation of m6A by a complex interplay of oxidizing and converting. Research has shown that FTO promoted metastatic spread in EC via HOXB13 mediated activation of WNT signaling¹⁸. In cervical cancer, FTO enhanced resistance to chemo- and radiotherapy through altered ß-catenin expression caused by m6A demethylation ²⁰. This might also be applicable to EC as FTO overexpression might contribute to the failure of radiation and chemotherapy leading to unfavorable clinical outcomes. Of note, FTO expression is closely related to weight gain and obesity^13,21. Both are established risk factors for EC development. Thus, FTO overexpression appears to promote EC development both, directly via WNT signaling, and indirectly by increasing of risk factors¹⁸. In our study, we confirmed the prognostic value of FTO expression at the protein level. FTO overexpression was significantly associated with a shortened OS. The data shown render FTO a promising anticancer therapeutic target. Research has shown that inhibition of FTO using MO-I-500, a small molecule inhibitor, effectively suppressed the growth and colony formation of triple negative breast cancer cells²². Recently, data on a more potent FTO inhibitor, namely FB23-2 was published. In the respective study, FB23-2 significantly inhibited AML progression in xenograft transplanted mice²³. Interestingly, there is data showing that FTO inhibitors display also anti-obesity effects in vivo and in vitro. The connection between obesity and cancer pathways via FTO seems to be regulated by mammalian target protein rapamycin (mTOR)²⁴. Entacapone and Epigallocatechin gallate (EGCG) showed in animal disease models additional to the anti-obesity effect a synergistically inhibition of cancer cell lines²⁵. However, the aforementioned inhibitors are still in an early preclinical phase but may improve EC therapy in the future.

ALKBH5 is the second demethylase involved in m6A modification. ALKBH5 upregulation was found to promote proliferation and invasion of EC cells by activating the IGF1R signaling pathway²⁶. In our analyses, enhanced ALKBH5 expression showed a trend towards a shorter OS but without reaching statistical significance. METTL3 and METTL14 are both responsible in installation of m6A. Whereas METTL14 mainly contributes to the stability of the methylation process, METTL3 is the most important component in catalyzing the transfer of methyl groups to adenine bases in RNA. For both writers, we obtained prognostic values regarding OS in our EC cohort. In pancreatic cancer, absence of METTL3 resulted in increased sensitivity to anticancer treatment, in particular to chemotherapy with gemcitabine, 5-fluoruracil, and platinum²⁷. Hence, METTL3 overexpression might lead to decreased susceptibility for platinum-based chemotherapy which is applied as first line treatment in advanced EC. In hepatocellular carcinoma (HCC), METTL14 was identified to be involved in the malignant progression of HCC by regulating m6A downstream targets, including cysteine sulfinic acid decarboxylase (CSAD), glutamic- oxaloacetic transaminase 2 (GOT2), and suppressor of cytokine signaling 2 (SOCS2)²⁸. Within our analyses, the ‘reader’ HNRNPC, was identified among the m6A enzymes significantly associated with poor survival. In the oncological context, sparse is known regarding the role of HNRNPC. However, elevated expression levels have been observed in HCC, glioblastoma, melanoma, and lung cancer²⁹. Recently, Wu et al. demonstrated suppression of tumor growth by knockdown of HNRNPC in breast cancer cells and a breast cancer xenograft model. These findings suggest a potential oncogene addiction regarding HNRNPC²⁹.

Overall, our study provides further evidence for involvement of m6A RNA modification in EC carcinogenesis. Overexpression of FTO, METTL3, METTL14, HNRNPA2B1, and HNRNPC in EC are associated with a poor clinical outcome. With regard to therapeutic implications, FTO appears to be an interesting target due to the connection of obesity and EC. However, further studies need to be performed to investigate in detail the biological functions and corresponding molecular mechanisms of m6a modifications in EC.

High expression levels of proteins involved in m⁶A modification are associated with a poor overall survival in patients with EC independent of clinicopathological parameters. Hence, our study provides further evidence for m⁶A to serve as a prognostic biomarker and promising target for new anticancer therapeutics in EC.

Funding

Damian J. Ralser is supported by the BONFOR program of the Medical Faculty of the University of Bonn (grant ID 2021-1A-14).Niklas Klümper is supported by junior research group funding by the BONFOR Program of the Medical Faculty of the University of Bonn (grant ID 2020-2A-12).

The others authors declare that no funds, grants or other support were received during the preparation of this manuscript.

Competing Interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Damian Ralser, Mateja Condic, Christian Staerk, and Thore Thiesler. The first draft of the manuscript was written by Damian Ralser and Mateja Condic and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability statement

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Ethics

The study was approved by the Ethics Committee of the University of Bonn (vote: 208/21).

Acknowledgments

The tissue specimens were collected within the framework of the Biobank of the University Hospital Bonn. We thank Susanne Steiner for technical support.

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Table 1

Clincopathological characteristics.

Endometrial cancer cohort N=65

Age (years)

Mean

Min-max

Histology

endometrioid

serous-papillary

undifferentiated

Follow-up (months)

Mean

Min-max

FIGO classification

IIIA

IIIB

unknown

Lymph node involvement

Yes

Unkown

Grading

64.9 ± 11.2

41.8 – 89.2

60 (92.3%)

3 (4.6%)

2 (3.1%)

70.7 ± 46.1

0-177

23 (35.3%)

18 (27.7%)

12 (18.5%)

6 (9.2%)

5 (7.7%)

1 (1.5%)

10 (15.4%)

23 (35.4%)

32 (49.2%)

8 (12.3%)

42 (64.6%)

15 (23.1%)

Table 2

Summary of analyzed proteins and their associations with overall survival. Estimated hazard ratios (HR) with 95% confidence intervals based on univariate Cox regression analyses; P-values for the group comparison (low vs. high expression) based on log-rank tests.

No competing interests reported.

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Comprehensive immunohistochemical analysis of N6-methyladenosine (m6A) writers, erasers, and readers in endometrial cancer

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