Mitochondrial autoimmunity and MNRR1 in breast carcinogenesis

Breast cancer (BC) is a leading malignancy in the United States with a very high incidence of metastatic disease [1]. National efforts to institute standard screening measures using annual screening mammography have led to enhanced early detection of BC, including a large number of slow growing breast tumors with relatively good prognosis [2]. However, aggressive BC is still a cause of significant mortality among women, with over 40,000 BC-associated deaths reported in 2017 in the United States [3], and the early ability to differentiate indolent from aggressive BC is still lacking [4]. Multiple studies have demonstrated that the sera from patients with BC and other solid tumors consistently contain a variety of autologous cellular antigens or tumor-associated antigens [4]. A plethora of autoantibodies is known to be present in BC serum; we recently reported that anti-mitochondrial antibodies (AMAs) constitute a distinctive feature of the autoantibody profile of BC sera and proposed that autoimmunity participates in breast carcinogenesis [5]. We further reported that 6 of the 13 mitochondrial proteins comprising the ETC are BC autoantigens [6].

In this work, we report the autoreactivity of two clones identified from cDNA of BC proteins encoding a sequence identical to the mitochondrial regulator MNRR1. The identification of the nDNA-encoded bi-organellar protein MNRR1, which functions in both the mitochondria and the nucleus, as a BC autoantigen offered proof of principle that AMAs in BC sera are the expression of mitochondrial autoimmunity. When localized in the mitochondria, MNRR1 binds to and activates cytochrome c oxidase [7, 8] whereas in the nucleus it functions as a transcriptional activator for genes harboring an 8-base pair DNA core of a conserved 13-bp element that responds maximally at 4% experimental oxygen concentration, and therefore is referred to as the oxygen responsive element [8‐10].

MNRR1 expression has previously been associated with survival prognosis in a number of cancer types including lung [11] and liver cancers [12]; consequently, we explored the possibility of a direct role for MNRR1 in BC. In this work we show that MNRR1 is a breast cancer autoantigen that directly participates in breast metastasis. The present data supports our hypothesis that mitochondrial autoimmunity as well as MNRR1 auto-reactivity are involved in breast carcinogenesis. We further propose that detection of autoantibodies against MNRR1 in the sera of BC patients but not in control non-cancer sera suggests that MNRR1, alone or in conjunction with a panel of other AMAs, can contribute to the early diagnosis of BC and potentially differentiate indolent from aggressive disease.

Immunoscreening of a T7 cDNA display library of BC proteins constructed with messenger RNA from established BC cell lines and biopanned with BC sera rich in AMAs led to the cloning of a large number of expressed sequence tags (ESTs), 184 of them representing unique gene products with significant immunochemical reactivity with BC sera. Of these unique gene products, 87 phages had sequences encoded by mtDNA that were highly associated with the diagnosis of IDC [6]. In that work, 6 of the 13 mtDNA encoded proteins of the ETC were identified as BC autoantigens, including 67 clones of ND5 and one clone each of ND4, ND4L, ND6, ND6, COXI, MT-ATP6, and 16S rRNA (MTRNR2, encoding humanin; NC_012920.1; Q8IVG9.1) [18] and a panel (Table 1 and unpublished) of nDNA-encoded mitochondrial proteins recognized as autoantigens by BC sera but not by non-cancer control sera. Another one of these antigens, not included in Table 1, is MNRR1, the subject of this report.

Immunoscreening of the T7 cDNA display library of proteins identified a 220-bp cDNA clone encoding a sequence identical to MNRR1. The results of the homology search for this cDNA sequence are shown (Fig. 1a; GenBank Accession #2095989 Seq1 MH071716). BLASTn analysis resulted in the identification of expression sequence tags reflecting mRNA transcript variants 1 and 2 of Homo sapiens MNRR1 (also known as CHCHD2), with sequence identity of 99% and an alignment score of 438 (E value e^− 120). BLASTx search of this transcript resulted in the identification of H. sapiens MNRR1 precursor isoform 1 (Fig. 1b), with associated sequence identity of 100% (E value 0.03). This clone was highly associated with the diagnosis of IDC of the breast by autoantigen microarray analysis (P < 0.0001).

The identification of MNRR1 cDNA in our phage BC library, the association of this phage with the diagnosis of IDC of the breast in the autoantigen microarray, the increased expression of this mitochondrial protein in histologically confirmed malignant breast tissue, and the correlation of MNRR1 expression with markers of metastatic BC, all suggest that this protein is important in the pathophysiology of BC and, in particular, its metastasis. For example, the levels of AKT3, a negative regulator of BC metastasis [21, 22], were found to be inversely proportional to MNRR1 levels. Furthermore, RNA-Seq data in HEK cells and proteomic data in BC cell lines, taken together, are also consistent with the role of MNRR1 as a key regulator of genes involved in an aggressive phenotype.

The participation of a regulator of mitochondrial respiration such as MNRR1 in breast metastatic disease is in agreement with previous work identifying an oxidative phenotype in metastatic BC cells [27, 29]. At least two specific features of mitochondria besides their efficient production of ATP have emerged. One is the need for a controlled but elevated level of reactive oxygen species (ROS) production, in part arising at mitochondrial complexes I and III [30]. We have reported that several subunits of complex I are targeted by AMAs [6]. The ROS produced are needed for cellular signaling for both growth and metastasis, for instance by increasing SRC/PYK2 protein kinase signaling [28]. We have noted that several subunits of complex I of the ETC are targeted by autoantibodies (Table 1). In addition, we have found autoreactivity to several nDNA-encoded mitochondrial proteins including PHB2 and HIGD1A, which have been implicated in the pathogenesis of BC, as well the exchange factor SGEF, which suggests abnormal Src signaling in breast carcinogenesis (Table 2; unpublished). An important conclusion is that MNRR1, in addition to being an activator of mitochondrial function, also promotes cell migration [31], suggesting that both its mitochondrial and its nuclear functions are involved in the development of BC metastasis.

Our previous work and the data on PGC-1α (Fig. 6c) show that MNRR1 promotes mitochondrial respiration [7, 8]. In addition, MNRR1 promotes other features of a metastatic phenotype. These include the import of MNRR1 itself, since the presence of autoantibodies to TIMM8 in BC sera (Table 2) suggests the possibility of a disturbance in protein import, the inhibition of apoptosis through the interaction of MNRR1 with Bcl-xL [32], preventing outer mitochondrial membrane permeabilization, and the promotion of cellular migration [31].

MNRR1 expression has also been linked to lung cancer [11] and to follicular carcinoma of the thyroid [33]. In addition, studies on the pathogenesis of hepatocellular carcinoma have suggested a role for the involvement of MNRR1 in the process of carcinogenesis, mediated through the interaction of cyclic adenosine monophosphate response element-binding protein (CREB) with the MNRR1 promoter, leading to an increased level of its expression [12]. In agreement with our proposal, proteomic analysis of mammosphere models of BC has demonstrated a high degree of MNRR1 upregulation in this system [34]. These findings suggest both the involvement of MNRR1 in breast carcinogenesis and a potential role as a biomarker of aggressive BC. In this work we report that autoreactivity to MNRR1 is highly associated with the diagnosis of IDC of the breast by autoantigen microarray analysis. We further show here that properties associated with carcinogenesis are directly regulated by MNRR1.

We have previously reported the detection of a distinctive anti-mitochondria antibody profile in invasive BC, but also in DCIS of the breast and in a small group of healthy women with suspicious mammography findings but without detected BC on biopsy, importantly suggesting that autoantibodies are formed during the pre-malignant stage [5]. Furthermore, we have reported AMAs targeting mtDNA-encoded components of the ETC, which may represent an expression of mitochondrial autoimmunity [6]. It is likely that MNRR1 autoreactivity, as well as the autoreactivity observed in other nDNA-encoded mitochondrial proteins as well as the AMAs targeting the components of the ETC ([6] and Table 1), are expressions of mitochondrial autoimmunity in BC. We have now shown that IHC using a specific antibody to MNRR1 can contribute to distinguish malignant from benign breast tissue. This is important because mammographic assessment of women undergoing annual screening mammography is performed using the Breast Imaging and Reporting Data System (BI-RADS) [35] wherein the standard of care for a suspicious mammography assessment (BI-RADS4) is to follow with breast biopsy [36]. Only about 20% of these women are found to have BC whereas in about 80% the pathological exam reveals BBD. These healthy women are usually reassured and recommended to continue annual or bi-annual routine mammography study. We suggest that autoantibodies to a panel of mitochondrial antibodies including MNRR1, together with positive IHC staining for this protein, and perhaps for other nDNA- and mtDNA-encoded mitochondrial antigens, on breast biopsies performed routinely on women having a BIRADS4 assessment might indicate that the subject is at a higher risk for BC than is suggested by a BBD diagnosis found at breast biopsy. Since our study indicates that MNRR1 participates in breast carcinogenesis, our findings strongly suggest the need for further studies to elucidate the contribution of MNRR1 and other nDNA- and mtDNA-encoded mitochondrial antigens to the diagnosis of early BC.

In the context of the spectrum of autoantibodies reported in BC sera [4‐6], autoreactivity to MNRR1 and other proteins targeted by autoantibodies have potential as early markers of BC metastasis. We propose that a panel of mtDNA- and nDNA-encoded mitochondrial antigens should be evaluated for utility in BC screening to increase early BC detection and perhaps to identify healthy women at higher risk for BC. Based on the studies on mitochondrial function in BC and the findings reported here, aggressive cancer cells shift to an oxidative metabolism during a metastatic change [28, 37]. An important feature in breast carcinogenesis is the need for TCA cycle-derived intermediates for synthesis of growth precursors. To this effect, some of the AMAs we found in BC sera target key enzymes involved in carbohydrate metabolism in the mitochondria such as GAPDH and GSTP1 (Table 2). Detection of AMAs in BC indicates an immune response triggered by the host against antigens developing in the cancerous cells. There is an established causal relationship between chronic inflammation and a number of solid tumors [38, 39]. In other solid tumors such as BC chronic inflammatory changes are often found although in the absence of a diagnosable inflammatory or infectious event. Since auto-immune damage is known to produce significant cellular damage and chronic inflammation in the RADs [40, 41], we have proposed that autoimmune damage to the breast and perhaps as well to other solid tumors, induces an inflammatory milieu likely to promote carcinogenesis via multiple regulatory functions in the mitochondria such as oxidative phosphorylation, ROS signaling, and one-carbon metabolism [6].

AKT is a signaling protein that has been shown to play a key role in cancer progression and metastasis [42]. Akt has three isoforms, AKT1, AKT2, and AKT3, each with a distinct function in cell physiology. Of the three isoforms, AKT3 was initially proposed to be involved in CNS development [43]. Newer studies have shown it to have an inhibitory effect on cancer growth [44]. In breast cancer cells, downregulation of AKT3 has also been shown to enhance metastasis [21, 22]. AKT3 was the most upregulated gene in the MNRR1-KO cells and may be one potential pathway for our observation of reduced cell migration. In addition, it also highlights the importance of identifying selective AKT inhibitors as a therapeutic in BC.

MNRR1 regulates multiple genes that function in cell migration required for cancer metastasis and is higher in cell lines derived from tumors previously shown to have enhanced metastatic potential. Since MNRR1 was identified as an autoantigen in breast carcinogenesis, and the other mitochondrial autoantigens are potentially downstream of MNRR1, the present data strongly support the role of MNRR1 and mitochondrial autoimmunity in breast carcinogenesis.

In this work we focus on both the direct participation of MNRR1 in breast carcinogenesis and the demonstration of autoreactivity to this protein in BC sera and its expression in BC tissue as an example of mitochondrial autoimmunity in BC. However, it is clear that MNRR1 is not the only mitochondrial protein involved in breast carcinogenesis since we found autoreactivity to a number of mtDNA- and nDNA-encoded mitochondrial proteins in BC sera (Tables 1 and 2) that suggest the widespread autoreactivity to mitochondrial antigens is the expression of autoimmunity in BC. Finally, the identification of MNRR1 in both cell lines and patient samples of aggressive BC suggests that it could serve as both a biomarker and a new drug target.