Deciphering mRNA-circRNA-miRNA axes and miRNA regulators in early breast cancer using a breast epithelial risk-progression 3D culture model

Abstract

Breast Cancer accounts for the highest cancer incidence and mortality rate in women worldwide [1]. In Lebanon, breast cancer is the most common malignancy, with the highest incidence in the world for women below 40 and a mean age at diagnosis 10 years younger than in developed countries [2]. These women have low prevalence of deleterious BRCA mutations and present with poor prognosis and aggressive phenotypes due to the lack of diagnostic methods at such an early age [3]. Early-onset breast cancer is increasing drastically worldwide [2, 4, 5], which called for identifying potential noninvasive biomarkers and active players for risk-assessment of breast cancer initiation and early detection actions. We therefore focused our study on the post-transcriptional gene regulators, microRNAs (miRNAs), and their “sponges”, circularRNAs (circRNAs), which have been shown to mediate cancerous phenotypes [6, 7]. CircRNAs are a class of endogenous RNAs that originate from RNA splicing and back ligation. miRNAs are small endogenous non-coding RNAs that repress translation. CircRNAs can sponge miRNAs by exhibiting endogenous competing binding sites (one to several) for each specific target miRNA. They covalently bind the mature miRNA, downregulating its expression, hence preventing it from binding its own target mRNAs and repressing translation [7, 8]. miRNAs and circRNAs exhibit unique dysregulation signatures in cancers, are stable and abundant in body fluids and act as attractive novel noninvasive cancer biomarkers. While, mRNA-circRNA-miRNAs axes have been characterized in breast cancer [9-15], axes involved in premalignant transitions that might explain and contribute to heightened risk of breast cancer initiation have not been reported.

The mammary gland development, differentiation and tumorigenesis is dependent on gap junction organization and proper communication, which is mediated by connexins (Cxs) [16-19]. Cx43, the focus of our previous and current research studies [7, 20, 21] plays essential roles during mammary gland development [22, 23] and differentiation [24] and acts as a tumor suppressor [20, 21, 25]. Its loss and mislocalization influence breast cancer initiation [26], progression [27] and is associated with markers of poor prognosis[25]. The nontumorigenic luminal human breast epithelial HMT-3522 S1 (S1) cell line, cultured under three-dimensional (3D) conditions, form a fully polarized epithelium with a central lumen and apicolateral localization of Cx43. S1 cells recapitulate normal human breast tissue architecture [26]. Silencing Cx43 expression in these nontumorigenic S1 cells via shRNA (Cx43-KO-S1) resulted in cell cycle entry, perturbed apical polarity, mitotic spindle misorientation and loss of lumen, causing cell multilayering and priming cells for enhanced motility and invasion [26, 27]. These features represent architectural and phenotypical premalignant mammary lesions, as seen in vivo [28], and increase the risk of breast cancer initiation, thus marking Cx43-KO-S1 as a pretumorigenic culture model. Therefore, this 3D risk-progression culture model was used to capture key pretumorigenic changes and cancer initiation phenotypes that might be triggered by miRNAs and circRNAs into heightening the risk of breast cancer development.

In order to understand post-transcriptional mechanisms that underlie heightened risk of breast cancer initiation, preliminary results from the Lebanese population that are notoriously at the highest risk of this early malignancy were essential for such investigations. Nassar et al. [29] previously identified 74 dysregulated miRNAs in microarrays from 45 invasive ductal carcinoma (IDC) versus 17 normal adjacent breast tissues from the Lebanese population. Forty-two percent of the patients were below the age of 40 and were categorized into early-stage breast cancers. Through a comprehensive literature review, fifteen tumor-associated miRNAs were uncovered here among these patient miRNAs to be involved in early events of breast tumorigenesis that contribute to loss of acinar morphogenesis. Next, circRNA microarrays and miRNA-sequencing from 3D acini of the pretumorigenic Cx43-KO-S1 cells compared to the nontumorigenic S1 counterparts were performed. The results revealed 121 circRNAs and 65 miRNAs that were significantly dysregulated in response to Cx43 silencing in the cultured epithelia. Focusing on the possible sponging activity of the validated circRNAs to their target miRNAs, cross-comparison was done to the actual miRNA datasets from the cultured epithelia and the patient validation cohort. The results revealed Cx43/hsa_circ_0077755/miR-182 as a biomarker signature axis for heightened-risk of breast cancer initiation. Moreover, its differential dysregulation patterns might predict prognosis along breast cancer initiation and progression.

We next aimed to study whether dysregulated miRNAs from the young patient cohort may increase the risk of breast cancer initiation by disrupting breast epithelial polarity, transitioning cells from phenotypically normal epithelium to premalignant phenotype. Thus, a comparative analysis between 65 miRNAs that were significantly dysregulated in response to Cx43 silencing in cultured epithelia and the 15 tumor-associated patient miRNAs involved in epithelial polarity was performed. The first chosen miRNA was miR-183, which was commonly up-regulated among the tumor-associated patient miRNAs involved in epithelia polarity pathways and was downstream of Cx43 loss. Moreover, miR-183 was the most up-regulated miRNA in early-stage Lebanese breast cancer patient cohort (and matched US patients) [29] and its up-regulation conferred with the increased risk of cancer progression in the 3D culture model. The second miRNA, miR-492, was chosen from the panel of up-regulated tumor-associated patient miRNAs that were involved in epithelia polarity pathways but were not downstream of Cx43 loss. Ectopic over-expression of both miR-183 and miR-492 in the non-neoplastic S1 cells (through pLenti-III-miR-GPF tagged vectors) resulted in the formation of larger acini in 3D cultures, devoid of lumen assembly, with disrupted epithelial polarity observed through mislocalization of ß-catenin and Scrib’s apico-lateral distribution and Cx43’s apical distribution. It also triggered enhanced proliferation and invasion capacity, hence recapitulating tumor-initiation phenotypes seen upon Cx43 loss [26, 27]. Tumor-initiation phenotypes attributed to miR-183 over-expression seem to be due to complete loss of Scrib in miR-183-S1 cells and mis-localization of the distribution of ß-catenin and Cx43 in the cultured epithelia. While loss of Scrib in miR-183-S1 cells seems to drive tumor-initiation phenotypes, its mis-localization was also reported to be sufficient in promoting tumorigenesis [30]. Therefore, tumor-initiation phenotypes attributed to miR-492 over-expression seem to be due to mislocalization of Scrib, ß-catenin and Cx43 in the cultured epithelia. Moreover, while miR-183 was found up-regulated in response to Cx43 loss, adding a direct link between Cx43 down-regulation and miR-183 up-regulation, Cx43 total mRNA levels were unchanged upon the over-expression of both miRNAs. Therefore, miR-183 and miR-492 over-expression might in part be affecting polarity disruption in a gap junctional dependent manner, through the mislocalization of Cx43 from apical membrane domains in aggregates formed in 3D culture, but not through directly down-regulating Cx43 expression. Although each miRNA is predicted to affect different downstream targets and pathways along breast cancer initiation, both miR-183 and miR-492 ectopic over-expression triggers enhanced proliferation and invasion and loss of apical polarity through Scrib’s loss/mis-localization and through altering the distribution of Cx43 and ß-catenin in the cultured epithelia.