Autophagy and senescence facilitate the development of antiestrogen resistance in ER positive breast cancer.

Estrogen receptor positive (ER+) breast cancer is the most common breast cancer diagnosed annually in the US with endocrine-based therapy as standard-of-care for this breast cancer subtype. Endocrine therapy includes treatment with antiestrogens, such as selective estrogen receptor modulators (SERMs), selective estrogen receptor downregulators (SERDs), and aromatase inhibitors (AIs). Despite the appreciable remission achievable with these treatments, a substantial cohort of women will experience primary tumor recurrence, subsequent metastasis, and eventual death due to their disease. In these cases, the breast cancer cells have become resistant to endocrine therapy, with endocrine resistance identified as the major obstacle to the medical oncologist and patient. To combat the development of endocrine resistance, the treatment options for ER+, HER2 negative breast cancer now include CDK4/6 inhibitors used as adjuvants to antiestrogen treatment. In addition to the dysregulated activity of CDK4/6, a plethora of genetic and biochemical mechanisms have been identified that contribute to endocrine resistance. These mechanisms, which have been identified by lab-based studies utilizing appropriate cell and animal models of breast cancer, and by clinical studies in which gene expression profiles identify candidate endocrine resistance genes, are the subject of this review. In addition, we will discuss molecular targeting strategies now utilized in conjunction with endocrine therapy to combat the development of resistance or target resistant breast cancer cells. Of approaches currently being explored to improve endocrine treatment efficacy and patient outcome, two adaptive cell survival mechanisms, autophagy, and "reversible" senescence, are considered molecular targets. Autophagy and/or senescence induction have been identified in response to most antiestrogen treatments currently being used for the treatment of ER+ breast cancer and are often induced in response to CDK4/6 inhibitors. Unfortunately, effective strategies to target these cell survival pathways have not yet been successfully developed. Thus, there is an urgent need for the continued interrogation of autophagy and "reversible" senescence in clinically relevant breast cancer models with the long-term goal of identifying new molecular targets for improved treatment of ER+ breast cancer.

Stress biology: Complexity and multifariousness in health and disease.

Preserving and regulating cellular homeostasis in the light of changing environmental conditions or developmental processes is of pivotal importance for single cellular and multicellular organisms alike. To counteract an imbalance in cellular homeostasis transcriptional programs evolved, called the heat shock response, unfolded protein response, and integrated stress response, that act cell-autonomously in most cells but in multicellular organisms are subjected to cell-nonautonomous regulation. These transcriptional programs downregulate the expression of most genes but increase the expression of heat shock genes, including genes encoding molecular chaperones and proteases, proteins involved in the repair of stress-induced damage to macromolecules and cellular structures. Sixty-one years after the discovery of the heat shock response by Ferruccio Ritossa, many aspects of stress biology are still enigmatic. Recent progress in the understanding of stress responses and molecular chaperones was reported at the 12th International Symposium on Heat Shock Proteins in Biology, Medicine and the Environment in the Old Town Alexandria, VA, USA from 28th to 31st of October 2023.

Targeted Replacement of HSF1 Phosphorylation Sites at S303/S307 with Alanine Residues in Mice Increases Cell Proliferation and Drug Resistance.

Mammalian heat shock factor HSF1 transcriptional activity is controlled by a multitude of phosphorylations that occur under physiological conditions or following exposure of cells to a variety of stresses. One set of HSF1 phosphorylation is on serine 303 and serine 307 (S303/S307). These HSF1 phosphorylation sites are known to repress its transcriptional activity. Here, we describe a knock-in mouse model where these two serine residues were replaced by alanine residues and have determined the impact of these mutations on cellular proliferation and drug resistance. Our previous study using this mouse model indicated the susceptibility of the mutant mice to become obese with age due to an increase in basal levels of heat shock proteins (HSPs) and chronic inflammation. Since HSF1 transcriptional activity is increased in many tumor types, this mouse model may be a useful tool for studies related to cellular transformation and cancer.

Nrf2 Drives Hepatocellular Carcinoma Progression through Acetyl-CoA-Mediated Metabolic and Epigenetic Regulatory Networks.

Correlations between the oxidative stress response and metabolic reprogramming have been observed during malignant tumor formation; however, the detailed mechanism remains elusive. The transcription factor Nrf2, a master regulator of the oxidative stress response, mediates metabolic reprogramming in multiple cancers. In a mouse model of hepatocellular carcinoma (HCC), through metabolic profiling, genome-wide gene expression, and chromatin structure analyses, we present new evidence showing that in addition to altering antioxidative stress response signaling, Nrf2 ablation impairs multiple metabolic pathways to reduce the generation of acetyl-CoA and suppress histone acetylation in tumors, but not in tumor-adjacent normal tissue. Nrf2 ablation and dysregulated histone acetylation impair transcription complex assembly on downstream target antioxidant and metabolic regulatory genes for expression regulation. Mechanistic studies indicate that the regulatory function of Nrf2 is low glucose dependent, the effect of which is demolished under energy refeeding. Together, our results implicate an unexpected effect of Nrf2 on acetyl-CoA generation, in addition to its classic antioxidative stress response regulatory activity, integrates metabolic and epigenetic programs to drive HCC progression. IMPLICATIONS: This study highlights that Nrf2 integrates metabolic and epigenetic regulatory networks to dictate tumor progression and that Nrf2 targeting is therapeutically exploitable in HCC treatment.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

GT198 Is a Target of Oncology Drugs and Anticancer Herbs.

Tumor angiogenesis is a hallmark of cancer. Therapeutic drug inhibitors targeting angiogenesis are clinically effective. We have previously identified GT198 (gene symbol PSMC3IP, also known as Hop2) as an oncoprotein that induces tumor angiogenesis in human cancers, including oral cancer. In this study, we show that the GT198 protein is a direct drug target of more than a dozen oncology drugs and several clinically successful anticancer herbs. GT198 is a DNA repair protein that binds to DNA. Using an in vitro DNA-binding assay, we tested the approved oncology drug set VII from the National Cancer Institute containing 129 oncology drugs. Identified GT198 inhibitors include but are not limited to mitoxantrone, doxorubicin, paclitaxel, etoposide, dactinomycin, and imatinib. Paclitaxel and etoposide have higher binding affinities, whereas doxorubicin has higher binding efficacy due to competitive inhibition. GT198 shares protein sequence homology with DNA topoisomerases, which are known drug targets, so that GT198 is likely a new drug target previously unrecognized. To seek more powerful GT198 inhibitors, we further tested several anticancer herbal extracts. The positive anticancer herbs with high affinity and high efficacy are all clinically successful ones, including allspice from Jamaica, Gleditsia sinensis or honey locust from China, and BIRM from Ecuador. Partial purification of allspice using an organic chemical approach demonstrated great feasibility of natural product purification, when the activity is monitored by the in vitro DNA-binding assay using GT198 as a target. Together, our study reveals GT198 as a new targeting mechanism for existing oncology drugs. The study also delivers an excellent drug target suitable for compound identification and natural product purification. In particular, this study opens an opportunity to rapidly identify drugs with high efficacy and low toxicity from nature.

Oncoprotein GT198 vaccination delays tumor growth in MMTV-PyMT mice.

Targeting early lesion in breast cancer is more therapeutically effective. We have previously identified an oncoprotein GT198 (PSMC3IP) in human breast cancer. Here we investigated GT198 in MMTV-PyMT mouse mammary gland tumors and found that GT198 is a shared early lesion in both species. Similar to human breast cancer even before a tumor appears, cytoplasmic GT198 is overexpressed in mouse tumor stroma including pericyte stem cells, descendent adipocytes, fibroblasts, and myoepithelial cells. Using recombinant GT198 protein as an antigen, we vaccinated MMTV-PyMT mice and found that the GT198 vaccine delayed mouse tumor growth and reduced lung metastasis. The antitumor effects were linearly correlated with vaccinated mouse serum titers of GT198 antibody, which recognized cell surface GT198 protein on viable tumor cells confirmed by FACS. Furthermore, GT198+ tumor cells isolated from MMTV-PyMT tumor induced faster tumor growths than GT198- cells when re-implanted into normal FVB/N mice. Together, this first study of GT198 vaccine in mouse showed its effectiveness in antitumor and anti-metastasis. The finding supports GT198 as a potential target in human immunotherapy since GT198 defect is shared in both human and mouse.

HSF1-Mediated Control of Cellular Energy Metabolism and mTORC1 Activation Drive Acute T-Cell Lymphoblastic Leukemia Progression.

Deregulated oncogenic signaling linked to PI3K/AKT and mTORC1 pathway activation is a hallmark of human T-cell acute leukemia (T-ALL) pathogenesis and contributes to leukemic cell resistance and adverse prognosis. Notably, although the multiagent chemotherapy of leukemia leads to a high rate of complete remission, options for salvage therapy for relapsed/refractory disease are limited due to the serious side effects of augmenting cytotoxic chemotherapy. We report that ablation of HSF1, a key transcriptional regulator of the chaperone response and cellular bioenergetics, from mouse T-ALL tumors driven by PTEN loss or human T-ALL cell lines, has significant therapeutic effects in reducing tumor burden and sensitizing malignant cell death. From a mechanistic perspective, the enhanced sensitivity of T-ALLs to HSF1 depletion resides in the reduced MAPK-ERK signaling and metabolic and ATP-producing capacity of malignant cells lacking HSF1 activity. Impaired mitochondrial ATP production and decreased intracellular amino acid content in HSF1-deficient T-ALL cells trigger an energy-saving adaptive response featured by attenuation of the mTORC1 activity, which is coregulated by ATP, and its downstream target proteins (p70S6K and 4E-BP). This leads to protein translation attenuation that diminishes oncogenic signals and malignant cell growth. Collectively, these metabolic alterations in the absence of HSF1 activity reveal cancer cell liabilities and have a profound negative impact on T-ALL progression. IMPLICATIONS: Targeting HSF1 and HSF1-dependent cancer-specific anabolic and protein homeostasis programs has a significant therapeutic potential for T-ALL and may prevent progression of relapsed/refractory disease.

The Molecular Chaperone Heat Shock Protein 70 Controls Liver Cancer Initiation and Progression by Regulating Adaptive DNA Damage and Mitogen-Activated Protein Kinase/Extracellular Signal-Regulated Kinase Signaling Pathways.

Delineating the mechanisms that drive hepatic injury and hepatocellular carcinoma (HCC) progression is critical for development of novel treatments for recurrent and advanced HCC but also for the development of diagnostic and preventive strategies. Heat shock protein 70 (HSP70) acts in concert with several cochaperones and nucleotide exchange factors and plays an essential role in protein quality control that increases survival by protecting cells against environmental stressors. Specifically, the HSP70-mediated response has been implicated in the pathogenesis of cancer, but the specific mechanisms by which HSP70 may support malignant cell transformation remains to be fully elucidated. Here, we show that genetic ablation of HSP70 markedly impairs HCC initiation and progression by distinct but overlapping pathways. This includes the potentiation of the carcinogen-induced DNA damage response, at the tumor initiation stage, to increase the p53-dependent surveillance response leading to the cell cycle exit or death of genomically damaged differentiated pericentral hepatocytes, and this may also prevent their conversion into more proliferating HCC progenitor cells. Subsequently, activation of a mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) negative feedback pathway diminishes oncogenic signals, thereby attenuating premalignant cell transformation and tumor progression. Modulation of HSP70 function may be a strategy for interfering with oncogenic signals driving liver cell transformation and tumor progression, thus providing an opportunity for human cancer control.

Modulation of Heat Shock Factor 1 Activity through Silencing of Ser303/Ser307 Phosphorylation Supports a Metabolic Program Leading to Age-Related Obesity and Insulin Resistance.

Activation of the adaptive response to cellular stress orchestrated by heat shock factor 1 (HSF1), which is an evolutionarily conserved transcriptional regulator of chaperone response and cellular bioenergetics in diverse model systems, is a central feature of organismal defense from environmental and cellular stress. HSF1 activity, induced by proteostatic, metabolic, and growth factor signals, is regulated by posttranscriptional modifications, yet the mechanisms that regulate HSF1 and particularly the functional significance of these modifications in modulating its biological activity in vivo remain unknown. HSF1 phosphorylation at both Ser303 (S303) and Ser307 (S307) has been shown to repress HSF1 transcriptional activity under normal physiological growth conditions. To determine the biological relevance of these HSF1 phosphorylation events, we generated a knock-in mouse model in which S303 and S307 were replaced with alanine (HSF1303A/307A). Our results confirmed that loss of phosphorylation in HSF1303A/307A cells and tissues increases protein stability but also markedly sensitizes HSF1 activation under normal and heat- or nutrient-induced stress conditions. Interestingly, the enhanced HSF1 activation in HSF1303A/307A mice activates a supportive metabolic program that aggravates the development of age-dependent obesity, fatty liver diseases, and insulin resistance. Thus, these findings highlight the importance of a posttranslational mechanism (through phosphorylation at S303 and S307 sites) of regulation of the HSF1-mediated transcriptional program that moderates the severity of nutrient-induced metabolic diseases.

Alteration of Tumor Metabolism by CD4+ T Cells Leads to TNF-α-Dependent Intensification of Oxidative Stress and Tumor Cell Death.

The inhibitory effects of cancer on T cell metabolism have been well established, but the metabolic impact of immunotherapy on tumor cells is poorly understood. Here, we developed a CD4+ T cell-based adoptive immunotherapy protocol that was curative for mice with implanted colorectal tumors. By conducting metabolic profiling on tumors, we show that adoptive immunotherapy profoundly altered tumor metabolism, resulting in glutathione depletion and accumulation of reactive oxygen species (ROS) in tumor cells. We further demonstrate that T cell-derived tumor necrosis factor alpha (TNF-α) can synergize with chemotherapy to intensify oxidative stress and tumor cell death in an NADPH (nicotinamide adenine dinucleotide phosphate hydrogen) oxidase-dependent manner. Reduction of oxidative stress, by preventing TNF-α-signaling in tumor cells or scavenging ROS, antagonized the therapeutic effects of adoptive immunotherapy. Conversely, provision of pro-oxidants after chemotherapy can partially recapitulate the antitumor effects of T cell transfer. These findings imply that reinforcing tumor oxidative stress represents an important mechanism underlying the efficacy of adoptive immunotherapy.

Targeted Deletion of Hsf1, 2, and 4 Genes in Mice.

Heat shock transcription factors (Hsfs) regulate transcription of heat shock proteins as well as other genes whose promoters contain heat shock elements (HSEs). There are at least five Hsfs in mammalian cells, Hsf1, Hsf2, Hsf3, Hsf4, and Hsfy (Wu, Annu Rev Cell Dev Biol 11:441-469, 1995; Morimoto, Genes Dev 12:3788-3796, 1998; Tessari et al., Mol Hum Repord 4:253-258, 2004; Fujimoto et al., Mol Biol Cell 21:106-116, 2010; Nakai et al., Mol Cell Biol 17:469-481, 1997; Sarge et al., Genes Dev 5:1902-1911, 1991). To understand the physiological roles of Hsf1, Hsf2, and Hsf4 in vivo, we generated knockout mouse lines for these factors (Zhang et al., J Cell Biochem 86:376-393, 2002; Wang et al., Genesis 36:48-61, 2003; Min et al., Genesis 40:205-217, 2004). Numbers of other laboratories have also generated Hsf1 (Xiao et al., EMBO J 18:5943-5952, 1999; Sugahara et al., Hear Res 182:88-96, 2003), Hsf2 (McMillan et al., Mol Cell Biol 22:8005-8014, 2002; Kallio et al., EMBO J 21:2591-2601, 2002), and Hsf4 (Fujimoto et al., EMBO J 23:4297-4306, 2004) knockout mouse models. In this chapter, we describe the design of the targeting vectors, the plasmids used, and the successful generation of mice lacking the individual genes. We also briefly describe what we have learned about the physiological functions of these genes in vivo.

Malignant pericytes expressing GT198 give rise to tumor cells through angiogenesis.

Angiogenesis promotes tumor development. Understanding the crucial factors regulating tumor angiogenesis may reveal new therapeutic targets. Human GT198 (PSMC3IP or Hop2) is an oncoprotein encoded by a DNA repair gene that is overexpressed in tumor stromal vasculature to stimulate the expression of angiogenic factors. Here we show that pericytes expressing GT198 give rise to tumor cells through angiogenesis. GT198+ pericytes and perivascular cells are commonly present in the stromal compartment of various human solid tumors and rodent xenograft tumor models. In human oral cancer, GT198+ pericytes proliferate into GT198+ tumor cells, which migrate into lymph nodes. Increased GT198 expression is associated with increased lymph node metastasis and decreased progression-free survival in oral cancer patients. In rat brain U-251 glioblastoma xenografts, GT198+ pericytes of human tumor origin encase endothelial cells of rat origin to form mosaic angiogenic blood vessels, and differentiate into pericyte-derived tumor cells. The net effect is continued production of glioblastoma tumor cells from malignant pericytes via angiogenesis. In addition, activation of GT198 induces the expression of VEGF and promotes tube formation in cultured U251 cells. Furthermore, vaccination using GT198 protein as an antigen in mouse xenograft of GL261 glioma delayed tumor growth and prolonged mouse survival. Together, these findings suggest that GT198-expressing malignant pericytes can give rise to tumor cells through angiogenesis, and serve as a potential source of cells for distant metastasis. Hence, the oncoprotein GT198 has the potential to be a new target in anti-angiogenic therapies in human cancer.

WITHDRAWN: Abrogation of heat shock factor 1 (Hsf1) phosphorylation deregulates its activity and lowers activation threshold, leading to obesity in mice.

This article has been withdrawn by the authors. During preparation of this manuscript, a number of errors occurred in the preparation/assembly of Figs 2D, 2E, S1C, S1E, and S4. The authors apologize for not acknowledging that Fig. 6E and 6J represented the same samples and therefore the ß-actin immunoblot was reused. These presentation errors do not impact the underlying scientific findings of the article and the article is being withdrawn so that a corrected manuscript can be submitted for publication. We are sorry for any problems or issues that this may have caused the scientific community.

The transcriptional regulator of the chaperone response HSF1 controls hepatic bioenergetics and protein homeostasis.

Metabolic energy reprogramming facilitates adaptations to a variety of stress conditions and cellular dysfunction, but how the energetic demands are monitored and met in response to physiological stimuli remains elusive. Our data support a model demonstrating that heat shock factor 1 (HSF1), a master transcriptional regulator of the chaperone response, has been coopted from its role as a critical protein quality-control regulator to having a central role in systemic energy sensing and for metabolic adaptation to nutrient availability. We found that in the absence of HSF1, levels of NAD+ and ATP are not efficiently sustained in hepatic cells, largely because of transcriptional repression of nicotinamide phosphoribosyltransferase in the NAD+ salvage pathway. Mechanistically, the defect in NAD+ and ATP synthesis linked to a loss of NAD+-dependent deacetylase activity, increased protein acetylation, and impaired mitochondrial integrity. Remarkably, the drop in ATP level caused by HSF1 loss invoked an adaptive response featuring the inhibition of energetically demanding processes, including gluconeogenesis, translation, and lipid synthesis. Our work identifies HSF1 as a central regulator of cellular bioenergetics and protein homeostasis that benefits malignant cell progression and exacerbates development of metabolic diseases.

GT198 Expression Defines Mutant Tumor Stroma in Human Breast Cancer.

Human breast cancer precursor cells remain to be elucidated. Using breast cancer gene product GT198 (PSMC3IP; alias TBPIP or Hop2) as a unique marker, we revealed the cellular identities of GT198 mutant cells in human breast tumor stroma. GT198 is a steroid hormone receptor coactivator and a crucial factor in DNA repair. Germline mutations in GT198 are present in breast and ovarian cancer families. Somatic mutations in GT198 are present in ovarian tumor stromal cells. Herein, we show that human breast tumor stromal cells carry GT198 somatic mutations and express cytoplasmic GT198 protein. GT198(+) stromal cells share vascular smooth muscle cell origin, including myoepithelial cells, adipocytes, capillary pericytes, and stromal fibroblasts. Frequent GT198 mutations are associated with GT198(+) tumor stroma but not with GT198(-) tumor cells. GT198(+) progenitor cells are mostly capillary pericytes. When tested in cultured cells, mutant GT198 induces vascular endothelial growth factor promoter, and potentially promotes angiogenesis and adipogenesis. Our results suggest that multiple lineages of breast tumor stromal cells are mutated in GT198. These findings imply the presence of mutant progenitors, whereas their descendants, carrying the same GT198 mutations, are collectively responsible for forming breast tumor microenvironment. GT198 expression is, therefore, a specific marker of mutant breast tumor stroma and has the potential to facilitate diagnosis and targeted treatment of human breast cancer.

Inhibitor of differentiation 1 transcription factor promotes metabolic reprogramming in hepatocellular carcinoma cells.

Reprograming of metabolism is one of the central hallmarks of cancer. The majority of cancer cells depend on high rates of glycolysis and glutaminolysis for their growth and survival. A number of oncogenes and tumor suppressors have been connected to the regulation of altered glucose and glutamine metabolism in cancer cells. For example, the oncogene c-Myc plays vital roles in cancer cell metabolic adaptation by directly regulating various genes that participate in aerobic glycolysis and glutaminolysis. Inhibitor of differentiation 1 (Id1) is a helix-loop-helix transcription factor that plays important roles in cell proliferation, differentiation, and cell fate determination. Overexpression of Id1 causes intestinal adenomas and thymic lymphomas in mice, suggesting that Id1 could function as an oncogene. Despite it being an oncogene, whether Id1 plays any prominent role in cancer cell metabolic reprograming is unknown. Here, we demonstrate that Id1 is strongly expressed in human and mouse liver tumors and in hepatocellular carcinoma (HCC) cell lines, whereas its expression is very low or undetectable in normal liver tissues. In HCC cells, Id1 expression is regulated by the MAPK/ERK pathway at the transcriptional level. Knockdown of Id1 suppressed aerobic glycolysis and glutaminolysis, suggesting that Id1 promotes a metabolic shift toward aerobic glycolysis. At the molecular level, Id1 mediates its metabolic effects by regulating the expression levels of c-Myc. Knockdown of Id1 resulted in down-regulation (∼75%) of c-Myc, whereas overexpression of Id1 strongly induced (3-fold) c-Myc levels. Interestingly, knockdown of c-Myc resulted in down-regulation (∼60%) of Id1, suggesting a positive feedback-loop regulatory mechanism between Id1 and c-Myc. Under anaerobic conditions, both Id1 and c-Myc are down-regulated (50-70%), and overexpression of oxygen-insensitive hypoxia-inducible factor 1α (Hif1α) or its downstream target Mxi1 resulted in a significant reduction of c-Myc and Id1 (∼70%), suggesting that Hif1α suppresses Id1 and c-Myc under anaerobic conditions via Mxi1. Together, our findings indicate a prominent novel role for Id1 in liver cancer cell metabolic adaptation.