Proteasome mapping reveals sexual dimorphism in tissue-specific sensitivity to protein aggregations.

Defects in the proteasome can result in pathological proteinopathies. However, the pathogenic role of sex- and tissue-specific sensitivity to proteotoxic stress remains elusive. Here, we map the proteasome activity across nine tissues, in male and female mice, and demonstrate strong sexual dimorphism in proteasome activity, where females have significantly higher activity in several tissues. Further, we report drastic differences in proteasome activity among tissues, independently of proteasome concentration, which are exacerbated under stress conditions. Sexual dimorphism in proteasome activity is confirmed in a SOD1 ALS mouse model, in which the spinal cord, a tissue with comparatively low proteasome activity, is severely affected. Our results offer mechanistic insight into tissue-specific sensitivities to proteostasis stress and into sex differences in the progression of neurodegenerative proteinopathies.

The Multi-Faced Role of PAPP-A in Post-Partum Breast Cancer: IGF-Signaling is Only the Beginning.

Insulin-like growth factor (IGF) signaling and control of local bioavailability of free IGF by the IGF binding proteins (IGFBP) are important regulators of both mammary development and breast cancer. A recent genome-wide association study (GWAS) identified small nucleotide polymorphisms that reduce the expression of IGFBP-5 as a risk factor of developing breast cancer. This observation suggests that genetic alterations leading to a decreased level of IGFBP-5 may also contribute to breast cancer. In the current review, we focus on Pregnancy-Associated Plasma Protein A (PAPP-A), a protease involved in the degradation of IGFBP-5. PAPP-A is overexpressed in the majority of breast cancers but its role in cancer has only begun to be explored. More specifically, this review aims at highlighting the role of post-partum involution in the oncogenic function of PAPP-A. Notably, we summarize recent studies indicating that PAPP-A plays a role not only in the degradation of IGFBP-5 but also in the deposition of collagen and activation of the collagen receptor discoidin 2 (DDR2) during post-partum involution. Finally, considering the immunosuppressive microenvironment of post-partum involution, we also discuss the unexpected finding made in Ewing Sarcoma that PAPP-A plays a role in immune evasion. While the immunosuppressive role of PAPP-A in breast cancer remains to be determined, collectively these studies highlight the multifaced role of PAPP-A in cancer that extends well beyond its effect on IGF-signaling.

SOD2 and the Mitochondrial UPR: Partners Regulating Cellular Phenotypic Transitions.

ATP and reactive oxygen species (ROS) are signaling molecules that control cellular function and phenotype. Mitochondria produce both ATP and ROS. Since the electrons needed to generate either ATP or ROS originate from NADH/FADH2, the mechanism through which electrons flow towards oxygen determines yields and whether ATP or ROS prevails. Alterations in the electron flow impact cells dramatically, such as by supporting specialization (which requires high ATP) or imposing dedifferentiation. High ROS, facilitated by enzymes such as superoxide dismutase 2 (SOD2) that enhance mitochondrial hydrogen peroxide (mtH2O2), are normally linked to dedifferentiation of somatic cells. Here we propose that combined high mtH2O2 and mitochondrial unfolded protein response (UPR(mt)) activation are essential for somatic dedifferentiation programs and the acquisition of stem-like properties in reparative processes and disease.

Cross talk between SOD1 and the mitochondrial UPR in cancer and neurodegeneration.

The mitochondrial unfolded protein response (UPRmt) is rapidly gaining attention. While the CHOP (ATF4/5) axis of the UPRmt was the first to be described, other axes have subsequently been reported. Validation of this complex pathway in C. elegans has been extensively studied. However, validation of the UPRmt in mouse models of disease known to implicate mitochondrial reprogramming or dysfunction, such as cancer and neurodegeneration, respectively, is only beginning to emerge. This review summarizes recent findings and highlights the major role of the superoxide dismutase SOD1 in the communication between the mitochondria and the nucleus in these settings. While SOD1 has mostly been studied in the context of familial amyotrophic lateral sclerosis (fALS), recent studies suggest that SOD1 may be a potentially important mediator of the UPRmt and converge to emphasize an increasingly vital role of SOD1 as a therapeutic target in cancer.

Parity predisposes breasts to the oncogenic action of PAPP-A and activation of the collagen receptor DDR2.

BACKGROUND: Women who had children at a young age (less than 25) show a reduced overall risk of breast cancer. However, epidemiological studies showed that for all other women, pregnancy increases the risk of breast cancer and the risk remains higher for decades. Further, even in women who had children at a young age, there is a transient increase risk that peaks 6 years after pregnancy. Women diagnosed with breast cancer following pregnancy show a higher rate of metastasis. Yet, the factors that increase the predisposition of post-partum breasts to more aggressive cancers remain unknown. Pregnancy-associated plasma protein A (PAPP-A) is a secreted protease that is overexpressed in more than 70% of breast cancers. However, PAPP-A is a collagen-dependent oncogene. We initiated this study to test the effect of PAPP-A on the predisposition of post-partum breasts. METHODS: We used PAPP-A mouse models for the analysis of its effect on virgin, involuting, or post-partum mammary glands. We performed second-harmonic generation microscopy for the analysis of collagen, defined tumor-associated collagen signature (TACS), the rate of mammary tumors, and the status of the collagen-DDR2-Snail axis of metastasis. We knockdown DDR2 by CRISPR and performed invasion assays. A transcriptomic approach was used to define a PAPP-A and parity-dependent genetic signature and assess its correlation with breast cancer recurrence in humans. RESULTS: We confirmed that post-partum mammary glands have a higher level of collagen than virgin glands and that this collagen is characterized by an anti-proliferative architecture. However, PAPP-A converts the anti-proliferative post-partum collagen into pro-tumorigenic collagen. We show that PAPP-A activates the collagen receptor DDR2 and metastasis. Further, deletion of DDR2 by CRISPR abolished the effect of PAPP-A on invasion. We defined a PAPP-A-driven genetic signature that identifies patients at higher risk of metastasis. CONCLUSIONS: These results support the notion that information about pregnancy may be critical in the prognosis of breast cancer as passage through a single pregnancy predisposes to the oncogenic action of PAPP-A. Our data indicate that history of pregnancy combined with the expression of PAPP-A-driven genetic signature may be useful to identify patients at higher risk of metastatic disease.

Sex specific activation of the ERα axis of the mitochondrial UPR (UPRmt) in the G93A-SOD1 mouse model of familial ALS.

The mitochondrial unfolded protein response (UPRmt) is a transcriptional program aimed at restoring proteostasis in mitochondria. Upregulation of mitochondrial matrix proteases and heat shock proteins was initially described. Soon thereafter, a distinct UPRmt induced by misfolded proteins in the mitochondrial intermembrane space (IMS) and mediated by the estrogen receptor alpha (ERα), was found to upregulate the proteasome and the IMS protease OMI. However, the IMS-UPRmt was never studied in a neurodegenerative disease in vivo. Thus, we investigated the IMS-UPRmt in the G93A-SOD1 mouse model of familial ALS, since mutant SOD1 is known to accumulate in the IMS of neural tissue and cause mitochondrial dysfunction. As the ERα is most active in females, we postulated that a differential involvement of the IMS-UPRmt could be linked to the longer lifespan of females in the G93A-SOD1 mouse. We found a significant sex difference in the IMS-UPRmt, because the spinal cords of female, but not male, G93A-SOD1 mice showed elevation of OMI and proteasome activity. Then, using a mouse in which G93A-SOD1 was selectively targeted to the IMS, we demonstrated that the IMS-UPRmt could be specifically initiated by mutant SOD1 localized in the IMS. Furthermore, we showed that, in the absence of ERα, G93A-SOD1 failed to activate OMI and the proteasome, confirming the ERα dependence of the response. Taken together, these results demonstrate the IMS-UPRmt activation in SOD1 familial ALS, and suggest that sex differences in the disease phenotype could be linked to differential activation of the ERα axis of the IMS-UPRmt.

From discovery of the CHOP axis and targeting ClpP to the identification of additional axes of the UPRmt driven by the estrogen receptor and SIRT3.

The mitochondrial UPR (UPRmt) is rapidly gaining attention. While most studies on the UPRmt have focused on its role in aging, emerging studies suggest an important role of the UPRmt in cancer. Further, several of the players of the UPRmt in mammalian cells have well reported roles in the maintenance of the organelle. The goal of this review is to emphasize aspects of the UPRmt that have been overlooked in the current literature, describe the role of specific players of the UPRmt in the biology of the mitochondria and highlight the intriguing possibility that targeting the UPRmt in cancer may be already within reach.

SOD2 to SOD1 switch in breast cancer.

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.

Inactivation of Omi/HtrA2 protease leads to the deregulation of mitochondrial Mulan E3 ubiquitin ligase and increased mitophagy.

Omi/HtrA2 is a nuclear encoded mitochondrial serine protease with dual and opposite functions that depend entirely on its subcellular localization. During apoptosis, Omi/HtrA2 is released into the cytoplasm where it participates in cell death. While confined in the inter-membrane space of the mitochondria, Omi/HtrA2 has a pro-survival function that may involve the regulation of protein quality control (PQC) and mitochondrial homeostasis. Loss of Omi/HtrA2's protease activity causes the neuromuscular disorder of the mnd2 (motor neuron degeneration 2) mutant mice. These mice develop multiple defects including neurodegeneration with parkinsonian features. Loss of Omi/HtrA2 in non-neuronal tissues has also been shown to cause premature aging. The normal function of Omi/HtrA2 in the mitochondria and how its deregulation causes neurodegeneration or premature aging are unknown. Here we report that the mitochondrial Mulan E3 ubiquitin ligase is a specific substrate of Omi/HtrA2. During exposure to H(2)O(2), Omi/HtrA2 degrades Mulan, and this regulation is lost in cells that carry the inactive protease. Furthermore, we show accumulation of Mulan protein in various tissues of mnd2 mice as well as in Omi/HtrA2(-/-) mouse embryonic fibroblasts (MEFs). This causes a significant decrease of mitofusin 2 (Mfn2) protein, and increased mitophagy. Our work describes a new stress-signaling pathway that is initiated in the mitochondria and involves the regulation of Mulan by Omi/HtrA2 protease. Deregulation of this pathway, as it occurs in mnd2 mutant mice, causes mitochondrial dysfunction and mitophagy, and could be responsible for the motor neuron disease and the premature aging phenotype observed in these animals.

Toca-1 is suppressed by p53 to limit breast cancer cell invasion and tumor metastasis.

INTRODUCTION: Transducer of Cdc42-dependent actin assembly-1 (Toca-1) recruits actin regulatory proteins to invadopodia, and promotes breast tumor metastasis. Since metastatic breast tumors frequently harbor mutations in the tumor suppressor p53, we tested whether p53 regulates Toca-1 expression. METHODS: Normal mammary epithelial cells (HBL-100, MCF10A) and breast cancer cell lines expressing wild-type (WT) p53 (DU4475, MTLn3) were treated with camptothecin or Nutlin-3 to stabilize p53 to test effects on Toca-1 mRNA and protein levels. Chromatin immunoprecipitation (ChIP) assays were performed to identify p53 binding site in Toca-1 gene. Stable silencing of p53 and Toca-1 were performed in MTLn3 cells to test effects on invadopodia and cell invasion in vitro, and tumor metastasis in vivo. RESULTS: We observed that breast cancer cell lines with mutant p53 have high levels of Toca-1 compared to those with WT p53. Stabilization of WT p53 led to further reduction in Toca-1 mRNA and protein levels in normal breast epithelial cells and breast cancer cells. ChIP assays revealed p53 binding within intron 2 of toca1, and reduced histone acetylation within its promoter region upon p53 upregulation or activation. Stable silencing of WT p53 in MTLn3 cells led to increased extracellular matrix degradation and cell invasion compared to control cells. Interestingly, the combined silencing of p53 and Toca-1 led to a partial rescue of these effects of p53 silencing in vitro and reduced lung metastases in mice. In human breast tumors, Toca-1 levels were high in subtypes with frequent p53 mutations, and high Toca-1 transcript levels correlated with increased risk of relapse. CONCLUSIONS: Based on these findings, we conclude that loss of p53 tumor suppressor function in breast cancers leads to upregulation of Toca-1, and results in enhanced risk of developing metastatic disease.

Idiosyncratic nature of lactation reveals link to breast cancer risk.

Breastfeeding protects against breast cancer in some women but not others, however the mechanism remains elusive. Lactation requires intense secretory activity of the endoplasmic reticulum (ER) for the production of milk proteins and ER-mitochondria contacts for lipid synthesis. We show that in female mice that share the same nuclear genome (BL/6) but differ in mitochondrial genomes (C57 or NZB), the biological processes engaged during lactation are entirely different at the sub-cellular organization and transcriptional levels resulting in anti-tumorigenic lactation in BL/6C57 females and pro-tumorigenic lactation in BL/6NZB females. Single cell sequencing identified a sub-population of cells, uniquely amplified during lactation in BL/6NZB females, which shares the genetic signature that characterizes post-partum breast cancer (PPBC) in humans relative to matched breast cancers in never pregnant women. Our data indicate that differences in ER and mitochondrial-stress responses during lactation between genotypes inadvertently leads to loss of p53 tumor suppressor function in BL/6NZB females allowing the expansion of the PPBC-like sub-population of cells. Overall, our data reveals the unexpected idiosyncratic nature of lactation and its impacts on the risk of the development of PPBC.

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.

Skp2B attenuates p53 function by inhibiting prohibitin.

The F-box protein Skp2 and its isoform Skp2B are both overexpressed in breast cancers. Skp2 alters the activity of p53 by inhibiting its interaction with p300 and by promoting p300 degradation. Here, we report that Skp2B also attenuates the activity of p53; however, this effect is independent of p300, suggesting that another mechanism might be involved. Prohibitin, a protein reported to activate p53, was isolated in a two-hybrid screen with the carboxy-terminal domain unique to Skp2B. We observed that prohibitin is a new substrate of Skp2B and that the degradation of prohibitin is responsible for the attenuated activity of p53 in cells overexpressing Skp2B. Furthermore, we show that the activity of p53 is reduced in the mammary glands of Skp2B transgenic mice. This study indicates that both Skp2 and Skp2B attenuate p53 activity through different pathways, suggesting that amplification of the Skp2 locus represents a powerful mechanism to attenuate p53 function in cancer.

Can THEM6 targeting stop resistance to prostate cancer treatment?

Prostate cancer (PCa) clinical management relies heavily on androgen-deprivation therapy (ADT). However, despite experiencing initial clinical benefit, patients getting ADT for non-resectable PCa eventually relapse and develop fatal castration-resistant PCa (CRPC). Multiple mechanisms of acquired resistance to treatment have been reported, including metabolic adaptation (Marine et al, 2020). Notably, activation of the endoplasmic reticulum (ER) unfolded protein response (UPR) has been associated with oncogenic transformation (Hart et al, 2012), tumor progression, metastasis dissemination, and resistance to therapy (Chen & Cubillos-Ruiz, 2021). Targeting different branches of ER UPR has been found to be an effective tool against aggressive PCa (Nguyen et al, 2018; Sheng et al, 2019). Therefore, a better understanding of these pathways may lead to the identification of novel drug targets.

Age alters the oncogenic trajectory toward luminal mammary tumors that activate unfolded proteins responses.

A major limitation in the use of mouse models in breast cancer research is that most mice develop estrogen receptor-alpha (ERα)-negative mammary tumors, while in humans, the majority of breast cancers are ERα-positive. Therefore, developing mouse models that best mimic the disease in humans is of fundamental need. Here, using an inducible MMTV-rtTA/TetO-NeuNT mouse model, we show that despite being driven by the same oncogene, mammary tumors in young mice are ERα-negative, while they are ERα-positive in aged mice. To further elucidate the mechanisms for this observation, we performed RNAseq analysis and identified genes that are uniquely expressed in aged female-derived mammary tumors. We found these genes to be involved in the activation of the ERα axis of the mitochondrial UPR and the ERα-mediated regulation of XBP-1s, a gene involved in the endoplasmic reticulum UPR. Collectively, our results indicate that aging alters the oncogenic trajectory towards the ERα-positive subtype of breast cancers, and that mammary tumors in aged mice are characterized by the upregulation of multiple UPR stress responses regulated by the ERα.

The portrait of liver cancer is shaped by mitochondrial genetics.

Cancer heterogeneity and evolution are not fully understood. Here, we show that mitochondrial DNA of the normal liver shapes tumor progression, histology, and immune environment prior to the acquisition of oncogenic mutation. Using conplastic mice, we show that mtDNA dictates the expression of the mitochondrial unfolded protein response (UPRmt) in the normal liver. Activation of oncogenic mutations in UPRmt-positive liver increases tumor incidence and histological heterogeneity. Further, in a subset of UPRmt-positive mice, invasive liver cancers develop. RNA sequencing (RNA-seq) analysis of the normal liver reveals that, in this subset, the PAPP-A/DDR2/SNAIL axis of invasion pre-exists along with elevated collagen. Since PAPP-A promotes immune evasion, we analyzed the immune signature and found that their livers are immunosuppressed. Further, the PAPP-A signature identifies the immune exhausted subset of hepatocellular carcinoma (HCC) in humans. Our data suggest that mtDNA of normal liver shapes the entire liver cancer portrait upon acquisition of oncogenic mutations.

Germline CAG repeat length of the androgen receptor and time to progression in patients with prostate cancer treated with androgen deprivation therapy.

OBJECTIVES: • Germline CAG repeat polymorphisms in the androgen receptor (AR-CAG) have been shown to influence the activity of the AR. • The purpose of the present study was to determine if AR-CAG repeat length correlates with time to progression on androgen deprivation therapy (ADT). PATIENTS AND METHODS: • Germline AR-CAG repeat lengths were determined in a cohort of 480 patients with recurrent or metastatic prostate cancer treated at a single tertiary care institution and correlated to time to progression (TTP) and overall survival. RESULTS: • There was no significant correlation between differences in the AR-CAG repeat lengths and TTP or overall survival in patients with prostate cancer receiving ADT. • AR-CAG repeat lengths did not significantly correlate with age, prostate-specific antigen (PSA), Gleason score or clinical stage at diagnosis. • In patients with metastatic disease, longer AR-CAG repeat lengths (>23 vs ≤23) were associated with a longer TTP on ADT, but this finding was of borderline significance (median TTP 18.3 vs 15.5 months, P = 0.09; adjusted HR = 0.76, 95% confidence interval = 0.54-1.09). CONCLUSIONS: • This is the largest published study to date investigating the association of germline AR-CAG repeat lengths and efficacy of ADT in prostate cancer. • Germline AR-CAG repeat lengths do not predict response to ADT.

Folding Mitochondrial-Mediated Cytosolic Proteostasis Into the Mitochondrial Unfolded Protein Response.

Several studies reported that mitochondrial stress induces cytosolic proteostasis. How mitochondrial stress activates proteostasis in the cytosol remains unclear. However, the cross-talk between the mitochondria and cytosolic proteostasis has far reaching implications for treatment of proteopathies including neurodegenerative diseases. This possibility appears within reach since selected drugs have begun to emerge as being able to stimulate mitochondrial-mediated cytosolic proteostasis. In this review, we focus on studies describing how mitochondrial stress activates proteostasis in the cytosol across multiple model organisms. A model is proposed linking mitochondrial-mediated regulation of cytosolic translation, folding capacity, ubiquitination, and proteasome degradation and autophagy as a multi layered control of cytosolic proteostasis that overlaps with the integrated stress response (ISR) and the mitochondrial unfolded protein response (UPRmt). By analogy to the conductor in an orchestra managing multiple instrumental sections into a dynamically integrated musical piece, the cross-talk between these signaling cascades places the mitochondria as a major conductor of cellular integrity.