Hsa-miR-3651 could serve as a novel predictor for in-breast recurrence via FRMD3

The breast cancer mortality rate in Europe is predicted to be 13.4 per 1,00,000 for 2020 [1]. Loco-regional control is a cornerstone for long-term cure. An analysis by the Early Breast Cancer Trialist Group (EBCTG) revealed a 4:1 relation between local relapse and breast cancer specific death [2]. Therapeutic advances in all disciplines related to breast conserving therapy (BCT) have led to low local relapse rates, with age being the most important predictor. The estimates for age adjusted annual in-breast recurrence are 0.4–0.7% for patients  > 50 years, 0.72–1.2% in the age group 41–50 years, and 0.72–2% for patients younger than 40 years, respectively [3]. As the prevention of local relapse is fundamental for long-term cancer free survival especially in younger patients [2], pre-therapeutic molecular tumor profiling is increasingly important.

MicroRNAs are small non-coding RNAs (ncRNA) of 20–25 nucleotides, which interfere in multiple (patho)physiological processes via transcriptional regulation. During the past 2 decades, they have emerged as a new class of predictive and prognostic markers in various types of cancer. As for breast cancer, Iorio suggested a miR-signature, describing a panel of de-regulated miRs correlated with known clinical and biological features such as hormonal receptor status, tumor size, lymph node status, vascular invasion, proliferation index and p53 [4]. Integrating of molecular markers in the clinical subtyping of breast cancer [5] opens up the way for ncRNA as a prediction tool in the future. The significance of microRNAs for local control (LC) could be demonstrated in three previous publications [6‐8]. While hsa-miR-3651 has been described in other cancer types such as head and neck [9, 10], ovarian [11], colorectal [12], liver [13] and lung [14], the current study is the first to demonstrate its relevance in breast cancer. Thus far, no data are extant on its potential target proteins, let alone a potential pathway which it could interfere in. Computational analyses suggested FERM domain containing protein 3 (FRMD3), a member of the protein 4.1 superfamily, as a putative downstream target, which is involved in cytoskeletal protein binding and seems to play a role in cell motility and invasiveness [15‐17].

The aim of the current study was to identify microRNAs that could predict in-breast recurrence in locoregionally highly invasive early stage breast cancers. This hypothesis-generating study is meant to enlarge the spectrum of tailored therapies for this disease in the future.

The current analysis revealed hsa-miR-3651 as a novel predictor for LC in early breast cancer. Additionally, FRMD3 was identified as a putative target that interferes with protein binding as well as cytoskeletal and cell membrane stability.

While hsa-miR-3651 has been described as a discriminatory marker in other cancer types, such as head and neck [9, 10], ovaries [11], colorectal [12], liver [13], lung [14] and nephroblastoma [30], the current analysis is the first in breast cancer. From the viewpoint of sample selection, the study by Tuncer can be regarded as related to ours, in as far as it also investigates hormone dependent tissue. [11]. The authors analyzed microRNA profiles in peripheral blood lymphocytes of monozygotic twins with ovarian cancer and healthy relatives. They also found up-regulated hsa-miR-3651 in cancer patients [11]. A study in oral squamous cell carcinoma (OSCC) comparing healthy versus tumor tissue showed elevated levels of hsa-miR-3651 in the latter [9, 10] with a fold change of 2.5. Likewise, the authors of this study detected a correlation between hsa-miR-3651 and clinical features that indicate loco-regional invasiveness, such as lymph node stage. In contrast, Wang et al. showed that the down-regulation of hsa-miR-3651 in esophageal cancer patients led to worse OS and DFS [31]. The authors could also demonstrate a correlation between clinical factors that add to loco-regional aggressiveness, such as T-, N-stage and tumor length and the expression levels of hsa-miR-3651, implying that this micro-RNA may exert its (patho)physiological function in a tissue dependent manner [31].

As for the role of hsa-miR-3651 and its potential interference in functional pathways, FRMD3 was identified as a putative target by microarray technology (Fig. 4) and validated in an independent set of patient using ddPCR (Figs. 5; supplementary file 5). This molecule, which belongs to the protein 4.1 superfamily, is characterized by the highly conserved membrane-association domain FERM [15]. Although comprehensive analyses about its functioning are not available to date, it is generally assumed that it links cytoskeletal structures, i.e., actin filaments, to membrane proteins, which enhances cellular stability and impacts loco-regional invasiveness [16, 17, 32] (GeneCodis 4.0 accessed February 2021, supplementary file 6). Apart from its role in metabolic diseases [33‐36], FRMD3 was reported to be focally expressed in hormone dependent tissue, i.e., adult ovary [37]. Additionally, in 58 NSCLC patient samples, Haase et al. found down-regulated FRMD3 in tumor compared to normal tissue [15]. In cell experiments the authors could show that it was able to induce apoptosis via the extrinsic, i.e., membrane dependent, pathway [15]. In contrast, reports on rectal [38, 39] and colon cancer [40] revealed that up-regulation of FRMD3 was associated with worse response to chemoradiotherapy. In two of the mentioned studies this molecule formed part of a multi-gene signature [39, 40]. In the third analysis, it was found to be the most up-regulated gene in nonresponders among 172 patients receiving cCRT [38]. Unfortunately, the authors did not verify their results in an independent set of individuals.

In summary, it can be said that in our study the de-regulation of hsa-miR-3651 measured as fold change is in the same range as in the above mentioned papers [9‐11]. With due caution it can be assumed that this microRNA plays a role in loco-regional invasiveness [31], which is, at least to some extent, corroborated by the current study. In this context, FRMD3, identified as a potential target, might play a crucial role [16, 17]. Discrepancies with other studies can be explained by differences in the choice of clinical material and methodology. Our analysis was conducted in breast cancer, while published data originate from other tissues [9, 10, 31]. The current study compares primary tumor samples of patients with and without relapse, while other analyses used normal tissue specimens as controls. Since a major technical challenge consists in improving the ”signal to noise ratio”, only specimen with at least 50% tumor content were selected, which is comparable to published literature [20]. Other groups chose a higher cancer cell cutoff [6] or used micro-dissection [18, 19] in order to maximize the tumor signal. Finally, the interplay between different types of miRNAs is an issue, therefore we focused on single microRNAs. Since clustered miRNAs, which make up approximately 25% of the whole miRnome, are located in close genomic vicinity their transcriptional efficacy is presumably higher than that of single microRNAs [21]. Hence, it does not seem counter-intuitive to assume that the latter are underrepresented in whole miRNome investigations. To account for this potential loss of information, miRNA families were excluded in the current analysis.

In spite of a predicted 94.5% probability for the interaction between hsa-miR-3651 and FRMD3, no mechanistic evidence on a cellular level is extant thus far. Secondly, the direct measurement of FRMD3 protein levels, which would have certainly strengthened our results, was impossible both for lack of a suitable antibody and the small number of cases in the protein validation phase. Our results, however, are coherent with the two-fold physiological functioning of a microRNA, which results in decreased mRNA levels of a target protein either by RISC-mediated dissection of the mRNA molecule based on a perfect miRNA-mRNA binding or delayed degradation by less exact match. Hence, changes in mRNA levels can be regarded as an indirect measure for protein concentrations. Additionally, an 8mer miR-mRNA binding site allows to postulate a close affinity between hsa-miR-3651 and FRMD3. A general disadvantage of molecular analyses in samples of a certain age is the degradation of the tissue, which could also be observed in the current study (supplementary files 2, 3, 7).

The current analysis demonstrates that hsa-miR-3651 may predict local control in early breast cancer via FRMD3. This hypothesis generating study provides a sound hypothesis to be tested in larger prospective studies, so that our results contribute to the framework of personalized medicine for this disease in the long run.