Luena Papa

SirT3 Regulates the Mitochondrial Unfolded Protein Response

ABSTRACT The mitochondria of cancer cells are characterized by elevated oxidative stress caused by reactive oxygen species (ROS). Such an elevation in ROS levels contributes to mitochondrial reprogramming and malignant transformation. However, high levels of ROS can cause irreversible damage to proteins, leading to their misfolding, mitochondrial stress, and ultimately cell death. Therefore, mechanisms to overcome mitochondrial stress are needed. The unfolded protein response (UPR) triggered by accumulation of misfolded proteins in the mitochondria (UPRmt) has been reported recently. So far, the UPRmt has been reported to involve the activation of CHOP and estrogen receptor alpha (ERα). The current study describes a novel role of the mitochondrial deacetylase SirT3 in the UPRmt. Our data reveal that SirT3 acts to orchestrate two pathways, the antioxidant machinery and mitophagy. Inhibition of SirT3 in cells undergoing proteotoxic stress severely impairs the mitochondrial network and results in cellular death. These observations suggest that SirT3 acts to sort moderately stressed from irreversibly damaged organelles. Since SirT3 is reported to act as a tumor suppressor during transformation, our findings reveal a dual role of SirT3. This novel role of SirT3 in established tumors represents an essential mechanism of adaptation of cancer cells to proteotoxic and mitochondrial stress.

Estrogen receptor mediates a distinct mitochondrial unfolded protein response

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2). Moreover, we demonstrate that the IMS stress-induced UPR culminates in increased proteasome activity. Given our previous report on a proteasome- and OMI-dependent checkpoint that limits the import of IMS proteins, the findings presented in this study suggest that this newly discovered UPR acts as a cytoprotective response to overcome IMS stress.

KLF6-SV1 Drives Breast Cancer Metastasis and Is Associated with Poor Survival

The KLF6-SV1 splice variant is associated with poor prognosis in early-stage human breast cancer and drives metastasis through the regulation of an EMT-like program in culture and in vivo. A New TWIST on Breast Cancer Sprawl Suburban sprawl is a complex, multistep process whereby “healthy” land becomes inundated with fast food chains, strip malls, and large parking lots. Yet, what drives sprawl is unclear—Do car-centric residential communities pop up close to businesses, or are the businesses merely opening where the consumers are? Cancer metastasis can be thought of as another type of sprawl, and although we can describe changes associated with metastasis, the drivers are equally unclear. Now, Hatami et al. provide insight into one of the potential drivers of breast cancer metastasis. The authors found that KLF6-SV1, which is a variant of a tumor suppressor gene, was associated with increased metastatic potential and poor survival in breast cancer patients. They then took their studies to the next step, trying to figure out how exactly KLF6-SV1 contributed to metastasis. Overexpressing KLF6-SV1 contributed to an epithelial-to-mesenchymal transition (EMT), which is thought to be important for cancer cells to leave the primary tumor. Indeed, inhibiting KLF6-SV1 returned these cells to a more epithelial (less metastatic) phenotype. Moreover, KLF6-SV1 alters the expression of TWIST1, which regulates EMT. Thus, KLF6-SV1 may be an early driver for metastasis in breast cancer patients. Metastasis is the major cause of cancer mortality. A more thorough understanding of the mechanisms driving this complex multistep process will aid in the identification and characterization of therapeutically targetable genetic drivers of disease progression. We demonstrate that KLF6-SV1, an oncogenic splice variant of the KLF6 tumor suppressor gene, is associated with increased metastatic potential and poor survival in a cohort of 671 lymph node–negative breast cancer patients. KLF6-SV1 overexpression in mammary epithelial cell lines resulted in an epithelial-to-mesenchymal–like transition and drove aggressive multiorgan metastatic disease in multiple in vivo models. Additionally, KLF6-SV1 loss-of-function studies demonstrated reversion to an epithelial and less invasive phenotype. Combined, these findings implicate KLF6-SV1 as a key driver of breast cancer metastasis that distinguishes between indolent and lethal early-stage disease and provides a potential therapeutic target for invasive breast cancer.

SOD2 to SOD1 Switch in Breast Cancer

Background: Cancer cells are characterized by elevated mitochondrial ROS. The dismutases SOD1 and SOD2 regulate ROS. Results: SOD2 is down-regulated following oncogenic activation in breast cancers. However, SOD1 is overexpressed, and its inhibition by LCS-1 leads to mitochondrial fragmentation. Conclusion: In the absence of SOD2, inhibition of SOD1 abolishes the integrity of the mitochondria. Significance: Our data suggest a SOD switch during transformation. Cancer cells are characterized by elevated levels of reactive oxygen species, which are produced mainly by the mitochondria. The dismutase SOD2 localizes in the matrix and is a major antioxidant. The activity of SOD2 is regulated by the deacetylase SIRT3. Recent studies indicated that SIRT3 is decreased in 87% of breast cancers, implying that the activity of SOD2 is compromised. The resulting elevation in reactive oxygen species was shown to be essential for the metabolic reprograming toward glycolysis. Here, we show that SOD2 itself is down-regulated in breast cancer cell lines. Further, activation of oncogenes, such as Ras, promotes the rapid down-regulation of SOD2. Because in the absence of SOD2, superoxide levels are elevated in the matrix, we reasoned that mechanisms must exist to retain low levels of superoxide in other cellular compartments especially in the intermembrane space of the mitochondrial to avoid irreversible damage. The dismutase SOD1 also acts as an antioxidant, but it localizes to the cytoplasm and the intermembrane space of the mitochondria. We report here that loss of SOD2 correlates with the overexpression of SOD1. Further, we show that mitochondrial SOD1 is the main dismutase activity in breast cancer cells but not in non-transformed cells. In addition, we show that the SOD1 inhibitor LCS-1 leads to a drastic fragmentation and swelling of the matrix, suggesting that in the absence of SOD2, SOD1 is required to maintain the integrity of the organelle. We propose that by analogy to the cadherin switch during epithelial-mesenchymal transition, cancer cells also undergo a SOD switch during transformation.

Bortezomib Enhances the Efficacy of Fulvestrant by Amplifying the Aggregation of the Estrogen Receptor, Which Leads to a Proapoptotic Unfolded Protein Response

Purpose: Fulvestrant is known to promote the degradation of the estrogen receptor (ER) in the nucleus. However, fulvestrant also promotes the aggregation of the newly synthesized ER in the cytoplasm. Accumulation of protein aggregates leads to cell death but this effect is limited as a result of their elimination by the proteasome. We tested whether combining fulvestrant with the proteasome inhibitor, bortezomib, could enhance the accumulation of ER aggregates and cause apoptotic cell death. Experimental Design: The rate of aggregation of the ER was monitored in ER+ breast cancer cells lines, T47D, ZR-75.1, BT474, MDA-MB-361, MCF-7, fulvestrant resistance MCF-7, and tamoxifen-resistant T47D-cyclin D1 cells. Activation of the unfolded protein response, apoptosis, and metabolic rate were also monitored in these cell lines following treatment with fulvestrant, bortezomib, or bortezomib in combination with fulvestrant. Results: We found that bortezomib enhances the fulvestrant-mediated aggregation of the ER in the cytoplasm without blocking the degradation of the ER in the nucleus. Further, these aggregates activate a sustained unfolded protein response leading to apoptotic cell death. Further, we show that the combination induced tumor regression in a breast cancer mouse model of tamoxifen resistance. Conclusions: Adding bortezomib to fulvestrant enhances its efficacy by taking advantage of the unique ability of fulvestrant to promote cytoplasmic aggregates of the ER. As this effect of fulvestrant is independent of the transcriptional activity of the ER, these results suggest that this novel combination may be effective in breast cancers that are ER+ but estrogen independent. Clin Cancer Res; 17(8); 2292–300. ©2011 AACR.

Selected mitochondrial DNA landscapes activate the SIRT3 axis of the UPRmt to promote metastasis

By causing mitochondrial DNA (mtDNA) mutations and oxidation of mitochondrial proteins, reactive oxygen species (ROS) leads to perturbations in mitochondrial proteostasis. Several studies have linked mtDNA mutations to metastasis of cancer cells but the nature of the mtDNA species involved remains unclear. Our data suggests that no common mtDNA mutation identifies metastatic cells; rather the metastatic potential of several ROS-generating mutations is largely determined by their mtDNA genomic landscapes, which can act either as an enhancer or repressor of metastasis. However, mtDNA landscapes of all metastatic cells are characterized by activation of the SIRT/FOXO/SOD2 axis of the mitochondrial unfolded protein response (UPRmt). The UPRmt promotes a complex transcription program ultimately increasing mitochondrial integrity and fitness in response to oxidative proteotoxic stress. Using SOD2 as a surrogate marker of the UPRmt, we found that in primary breast cancers, SOD2 is significantly increased in metastatic lesions. We propose that the ability of selected mtDNA species to activate the UPRmt is a process that is exploited by cancer cells to maintain mitochondrial fitness and facilitate metastasis.

Abstract 1121: ER-negative breast cancer survive mitochondrial IMS-stress via SIRT3 activation

Mitochondrial dysfunction is central in carcinogenesis and has been associated with elevated levels of Reactive Oxygen Species (ROS). While moderate levels of ROS activate signaling cascade promoting tumor progression, high levels of ROS can lead to cell death. We previously demonstrated that Estrogen Receptor (ER)-positive breast cancer cells activate ER to overcome the mitochondrial impairment and reduce high levels of ROS induced by stress in the inter-mitochondrial membrane space (IMS). The ultimate outcome of ER activation is induction of a novel Unfolded Protein Response (UPR) that involves up-regulation of NRF-1, a major regulator of mitochondrial biogenesis as well as induction of OMI and proteasome, two critical elements of protein quality control required to monitor the quality of IMS proteins. In the present work, we have evidence that ER-negative breast cancer cells trigger a distinct mechanism to cope up with IMS-stress. Due to the lack of ER, the stress from the IMS is extended into the matrix of mitochondria leading to activation of the matrix deacetylase, SIRT3. SIRT3 is required to increase the antioxidant capacity by up-regulating the antioxidant enzyme, MnSOD and therefore reducing ROS. In addition, our results reveal that SIRT3 is essential for removal of the irreversible impaired mitochondria through mitophagy. Overall, our data indicate that ER-positive and ER-negative breast cancer induce different mechanism to conquer mitochondrial malfunction coupled with ROS overproduction and maintain their cellular integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1121. doi:1538-7445.AM2012-1121

Abstract 5314: An oncogenic splice variant of the KLF6 tumor suppressor gene is associated with poor survival and is a potent driver of breast cancer metastasis

Breast cancer is a leading causes of cancer death in women worldwide. It is a heterogeneous and genetically complex disease has left a significant proportion of patients with inadequate treatment options. Since metastasis and drug resistance pose significant challengea in breast cancer treatment, elucidating the mechanisms of these processes is critical for understanding the key drivers of disease progression and for the development of targeted therapies. Accumulating evidence from our laboratory and other groups suggest that the tumor suppressor gene, KLF6, and its oncogenic splice variant, KLF6-SV1, play a role in breast cancer progression, dissemination, and chemoresistance. Here we demonstrate that in multiple independent clinical cohorts of over 1200 breast cancer patients with defined clinical outcome, high KLF6-SV1 mRNA levels in the primary tumor associated with poor survival and disease recurrence. Specifically, upregulated KLF6-SV1 expression in the primary tumors correlated with poor survival independent of disease stage and grade. Thus, we hypothesized that KLF6-SV1 is an early driver/molecular determinant of invasive breast cancer. In order to investigate the functional/biological relevance of KLF6-SV1 in breast cancer development and progression, we performed a series of experiments using retroviral-based overexpression in multiple non-tumorigenic, tumorigenic, and metastatic breast cancer cell lines. We demonstrated that overexpression of KLF6-SV1 increased mesenchymal marker gene expression, cellular survival, invasion, as well as the migratory potential of KLF6-SV1 transduced cells. Interestingly, KLF6-SV1 did not increase growth rate of these mammary epithelial cell lines, similar to the clinical data which showed an absence of a correlation between KLF6-SV1 expression and primary tumor size. In a 3D model KLF6-SV1 upregulation disrupted mammary acinar morphogenesis promoting complex multiacinar structures. To assay whether the KLF6-SV1-induced EMT phenotype conferred increased metastatic potential in vivo, we injected tumorigenic cells expressing high levels of KLF6-SV1 subcutaneously into immunodeficient mice. Strikingly, KLF6-SV1 overexpression alone drove the entire metastatic cascade resulting in dissemination to many organs including the liver, kidney, heart, lung, and spleen. Furthermore, KLF6-SV1 overexpression in a metastatic breast cancer cell line increased metastasis to the lungs and liver in an orthotopic model of the disease. This is consistent with our clinical data in which high KLF6-SV1 expression was correlated with decreased overall survival and metastasis free survival. Together these findings suggest a role for the KLF6-SV1 splice variant as a driver of breast cancer metastasis and validate its potential utility as a novel biomarker and therapeutic target for breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5314. doi:1538-7445.AM2012-5314

Abstract 2096: Estrogen receptor alpha activation mediates a novel mitochondrial unfolded protein response

Unfolded protein responses (UPR) of the endoplasmic-reticulum and of the matrix of the mitochondria have been described. Our data indicates that accumulation of proteins in the inter-membrane space of the mitochondria (IMS) activates a distinct UPR from that induced by stress in the mitochondrial matrix. We show that upon IMS-stress, cells activate the Estrogen Receptor (ERα) in a ligand independent fashion through ROS overproduction and AKT phosphorylation. ERα activation further triggers the induction of the mitochondrial regulator NRF-1 and the IMS protease Omi. In addition, IMS stress leads to an acceleration in the trypsin-like activity of the proteasome. Since we reported a proteasome and Omi-dependent checkpoint that limits the import of IMS proteins, these results suggest that this novel UPR acts as cyto-protective responses to overcome IMS-stress. Indeed, we show that whereas ERα is required for activation of these cyto-protective responses, its ligand-independent activation is necessary to prevent IMS stress-induced collapse in the mitochondrial potential. Notably, our result show that upon mitochondrial dysfunction, cells trigger the retrograde pathway involving the activation of ERα to facilitate the recovery of the physiological functions of the mitochondria and maintain their integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2096. doi:10.1158/1538-7445.AM2011-2096