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In most human cancers, somatic mutations have been identified in the mtDNA; however, their significance remains unclear. We recently discovered that NMuMG mouse mammary epithelial cells, when deprived of mitochondria or following inhibition of respiratory activity, undergo epithelial morphological disruption accompanied with irregular edging of E-cadherin, the appearance of actin stress fibers, and an altered gene expression profile. In this study, using the mtDNA-less pseudo ρ0 cells obtained from NMuMG mouse mammary epithelial cells, we examined the roles of two mitochondrial stress-associated transcription factors, cAMP-responsive element-binding protein (CREB) and C/EBP homologous protein-10 (CHOP), in the disorganization of epithelial phenotypes. We found that the expression of matrix metalloproteinase-13 and that of GADD45A, SNAIL and integrin α1 in the ρ0 cells were regulated by CHOP and CREB, respectively. Of note, knockdown and pharmacological inhibition of CREB ameliorated the disrupted epithelial morphology. It is interesting to note that the expression of high mobility group AT-hook 2 (HMGA2), a non-histone chromatin protein implicated in malignant neoplasms, was increased at the protein level through the CREB pathway. Here, we reveal how the activation of the CREB/HMGA2 pathway is implicated in the repression of integrin α1 expression in HepG2 human cancer cells, highlighting the importance of the CREB/HMGA2 pathway in malignant transformation associated with mitochondrial dysfunction, thereby raising the possibility that the pathway indirectly interferes with the cell–cell adhesion structure by influencing the cell–extracellular matrix adhesion status. Overall, the data suggest that mitochondrial dysfunction potentially contributes to neoplastic transformation of epithelial cells through the activation of these transcriptional pathways.
Over the past decades, a range of somatic mutations and depletions have been identified in the mtDNA in most primary human cancers.[1-3] However, the relationship between mtDNA instability and neoplastic cell development remains unclear. Recent studies with regard to mtDNA heteroplasmy in cancers have suggested that somatic mutations in mtDNA are enriched during tumorigenic processes, implying that they confer a selective advantage for the survival and growth of pre-neoplastic cells.[4-6] In other studies, several mutations in the mtDNA regions encoding polypeptides for the respiration and oxidative phosphorylation chain have been suggested to actively contribute to tumor progression and metastasis.[7, 8] It is noteworthy that all such mutations are associated with increased levels of reactive oxygen species (ROS), suggesting the involvement of ROS in the development of malignant phenotypes.
Somatic mutations in mtDNA are found not only in the protein-coding regions but also in the non-coding regions. In human tumors, point mutations are frequently observed (39.7% of the cancerous tissues examined) in the D-loop (non-coding) region. Mutations in this region, which contains the important regulatory sequences for transcription and replication initiation, hypothetically affect the copy number and/or gene expression of the mitochondrial genome. One study supporting this assumption reports a decrease in mtDNA copy number in 17 hepatocellular carcinoma (HCC) cases out of 24 cases with somatic mutation(s) in the D-loop region. Of note, instability in the mtDNA D-loop region leading to decreased copy number has been suggested to be involved in human carcinogenesis. In most cases of HCC and gastric cancers, carcinogenesis is accompanied with an alteration in mitochondrial biogenesis and a repression of mtDNA replication.[3, 10, 11] However, the effects of repression of mitochondrial biogenesis on tumorigenesis are poorly understood. The involvement of ROS in these processes remains an open question.
The present study examines the impact of decreased mitochondrial function on epithelial phenotypes associated with malignant transformations. We used the mtDNA-less pseudo ρ0 cells with decreased mitochondrial function. The ρ0 state was attained by inhibiting mtDNA replication and transcription using ethidium bromide (EtBr) rather than by D-loop mutations interfering with mtDNA replication and transcription. We obtained the ρ0 cells from NMuMG mouse mammary epithelial cells (NMuMG cells) and assessed their morphology and gene expression. In the ρ0 state, the typical cobblestone-like epithelial morphology was disrupted, resulting in irregular cell–cell junctions. We also observed some alterations in gene expression, including alterations in integrin α1 (ITGA1) expression. In conclusion, we demonstrated the importance of the two mitochondrial stress-associated transcription factors, particularly that of cAMP-responsive element-binding protein (CREB), in the disorganization of epithelial morphology.
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In general, lesions in the mtDNA coding or non-coding regions result in deficiencies in the respiratory chain. In a defective respiratory chain, aberrant ROS production often occurs as a result of leakage of electrons to oxygen, and, consequently, a catastrophic cycle of respiratory function dysregulation is created because of additional mutations caused by ROS. During such a cycle, the cells are continuously exposed to ROS and are either at a risk of developing oncogenic somatic mutations, thereby activating the oncogenic pathways, or are at risk of increased genome instability. Thus, the aberrant production of ROS possibly accounts for some of the effects of mtDNA mutations on tumorigenesis, especially in the initial stages.
In the final phase of respiratory chain deficiency, however, mitochondria are assumed to be completely deprived of their ROS-producing capability, regardless of the formation of the catastrophic cycle. In this phase, the impact of ROS and their signals presumably subsides. Instead, stress signals are likely to be elicited due to the sensing of the loss of normal mitochondrial functions. A recent study described such a stress response to mitochondrial dysfunction that was mediated by stress-inducible transcription factors, including CHOP and CREB. Hypoxia-inducible factor-1 α signaling, which requires mitochondria-derived ROS, appears to be dormant in the ρ0 cells.
In the present study, we explored the potential contribution of mitochondrial dysfunction in tumorigenesis as a stress modulating intracellular signaling during the later stages of the process. It is noteworthy that the incidence of somatic mutations in the D-loop of mtDNA is increased in late stage rather than early stage cancers. At the onset of neoplastic transformation in cells harboring mtDNA mutation(s), ROS are expected to be the dominant mutagens. However, we reasoned that in the resulting pre-neoplastic cells, stress signaling activated by decreased mitochondrial function would play an increased role in tumorigenic progression. In the present study, we observed changes in cell–cell junction structures as well as altered gene expression in the ρ0 cells, and characterized the roles of the CHOP and CREB mitochondrial stress mediators in altering the cellular phenotypes. This is the first report to evaluate the impact of mitochondrial dysfunction on epithelial morphology; cells of mesenchymal origin and fully transformed cancer cells have been studied previously.[16, 17, 23]
The mechanisms by which CREB and CHOP are upregulated under ρ0 conditions remain an open question. Based on our previous study, ROS are unlikely to be involved in signaling. Indeed, the phosphorylation of CREB was insensitive to an antioxidant, N-acetylcysteine (Motoko Shibanuma, unpublished data, 2012). The involvement of an unfolded protein response, which is a major stress inducing CHOP, is also unlikely. Instead, perturbed intracellular calcium distribution is the most likely mechanism. Mitochondria are implicated in intracellular calcium storage and homeostasis together with the endoplasmic reticulum (ER). In addition, they are physically associated, and the transport of calcium from one organelle to the other is highly efficient. In a previous report, ER calcium depletion and the consequent increase in the cytosolic fraction were shown to, respectively, induce CHOP and activate CREB. The inhibition of CREB phosphorylation by a CaM kinase inhibitor supports a similar involvement of calcium signaling in ρ0 cells (Fig. 5d).
Our findings revealed the critical role of CREB in the disorganization of epithelial phenotypes because of decreased mitochondrial functioning. The detailed mechanisms whereby the architecture of the cell–cell junctions of the ρ0 cells was disorganized downstream in the CREB pathway remain unclear. ATP depletion has been associated with disordered cell–cell adhesion. The upregulation of the SNAIL family members SNAIL, ZEB2 and SLUG may have contributed to this process through the induction of the epithelial–mesenchymal transition (EMT), as has been reported in some cases of adenocarcinoma. However, E-cadherin, an EMT regulator target, remained highly expressed, and so activation of EMT was unlikely in the ρ0 state. In other studies, depletion of mtDNA has also been associated with tumorigenic phenotypes, although, again, the detailed mechanisms remain unclear.[27-32]
Unexpectedly and interestingly, our study revealed a close relationship between CREB and HMGA2, namely, CREB regulation of HMGA2 expression, implying the existence of a novel transcriptional network organized by CREB and downstream HMGA2. HMGA2 is an oncofetal protein that is frequently amplified, rearranged and overexpressed in multiple human cancers,[18, 19] and it is causally related to neoplastic cell transformation.[33, 34] In this study, we originally identified ITGA1 as a potential transcriptional target of HMGA2. Given the possible interplay between cell–cell and cell–ECM adhesions, it is likely that the disorganized epithelial morphology that arises following mitochondrial dysfunction stems from a change in ITGA1 expression downstream in the CREB/HMGA2 cascade, which indirectly interferes with cell–cell adhesion structures by influencing cell–ECM adhesion status. Chen et al. report that ITGA1 serves as a negative regulator of epidermal growth factor receptor (EGFR) activity. This observation raises another interesting possibility that EGFR signaling, augmented by the repression of ITGA1 expression, is a primary cause of the disorganized epithelial structures in the ρ0 cells. Future studies should address the roles of the CREB/HMGA2 pathway in neoplastic transformation in more detail. The hypothetical contribution of mitochondrial dysfunction to malignant transformation in epithelial cells is illustrated in Figure 6(c), highlighting the roles of the CREB/HMGA2 pathway and the CHOP-mediated transcriptional cascade.
We suggest that the CREB/HMGA2 pathway operates not only in the ρ0 model but also in HepG2 human cancer cells. However, we also noticed a difference between HepG2 and NMuMG ρ0 model cells. For example, activation of the CHOP pathway was not detected in the human cancer cell lines studied (data not shown). Moreover, SNAIL and ZEB2 were not regulated by the CREB/HMGA2 pathway in HepG2 cells. Given that HMGA2 is an architectural nuclear factor modulating transcription through interaction with DNA and/or canonical transcription factors, the difference may be ascribed to the profiles of the transcription factors present in cells.
Interestingly, among cancerous tissues, HCC is characterized by a high number of mutations in the D-loop (42.6% of HCC carry the mutations) and a decreased mtDNA copy number (57.4%). Accordingly, the CREB/HMGA2 pathway deserves further research as a potential therapeutic target against HCC and other cancers that feature activated mitochondrial stress signaling.