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Background: Prostate cancer is a heterogenous disease, but once it becomes metastatic it eventually becomes treatment resistant. One mechanism of resistance to AR-targeting therapy is lineage plasticity, where the tumor undergoes a transformation to an AR-indifferent phenotype, most studied in the context of neuroendocrine prostate cancer (NEPC). However, activation of additional de- or trans-differentiation programs, including a gastrointestinal (GI) gene expression program, has been suggested as an alternative method of resistance. In this study, we explored the previously identified GI prostate cancer phenotype (PCa-GI) in a large cohort of metastatic castration-resistant prostate cancer (mCRPC) patient biopsy samples. Methods: We analyzed a dataset of 634 mCRPC samples with batch effect corrected gene expression data from the West Coast Dream Team (WCDT), the East Coast Dream Team (ECDT), the Fred Hutchinson Cancer Research Center (FHCRC) and the Weill Cornell Medical center (WCM). Survival data was available from the WCDT and ECDT cohorts. We calculated a gene expression GI score using the sum of z-scores of genes from a published set of PCa-GI-defining genes (N=38). Survival analysis was performed using the Kaplan-Meier method and Cox proportional hazards regression with endpoint overall survival from time of biopsy to death of any cause. Results: We found that the PCa-GI score had a bimodal distribution, identifying a distinct set of tumors with an activated GI expression pattern. Approximately 35% of samples were classified as PCa-GI high, which was concordant with prior reports. Liver metastases had the highest median score but after excluding liver samples, 29% of the remaining samples were still classified as PCa-GI high, suggesting a distinct phenotype not exclusive to liver metastases. No correlation was observed between GI score and proliferation, AR signaling, or NEPC scores. Furthermore, the PCa-GI score was not associated with genomic alterations in AR, FOXA1, RB1, TP53 or PTEN. However, tumors with MYC amplifications showed significantly higher GI scores (p=0.0001). Patients with PCa-GI tumors had a shorter survival (HR=1.5 [1.1-2.1], p=0.02), but this result was not significant after adjusting for the liver as metastatic site (HR=1.2 [0.82-1.7], p=0.35). Patients with PCa-GI low samples had a better outcome after androgen receptor signaling inhibitors (ASI, abiraterone or enzalutamide) than other therapies (HR=0.37 [0.22-0.61], p=0.0001) while the benefit of ASI was smaller and non-significant for PCa-GI high samples (HR=0.55 [0.29-1.1], p=0.07). A differential pathway analysis identified FOXA2 signaling to be upregulated PCa-GI high tumors (FDR = 3.7 × 10-13). Conclusions: The PCa-GI phenotype is prevalent in clinical mCRPC samples and may represent a distinct biological entity. PCa-GI tumors may respond less to ASI and could offer a strategy to study novel therapeutic targets.
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Histopathologic diagnosis and classification of cancer plays a critical role in guiding treatment. Advances in next-generation sequencing have ushered in new complementary molecular frameworks. However, existing approaches do not independently assess both site-of-origin (e.g. prostate) and lineage (e.g. adenocarcinoma) and have minimal validation in metastatic disease, where classification is more difficult. Utilizing gradient-boosted machine learning, we developed ATLAS, a pair of separate AI Tumor Lineage and Site-of-origin models from RNA expression data on 8249 tumor samples. We assessed performance independently in 10,376 total tumor samples, including 1490 metastatic samples, achieving an accuracy of 91.4% for cancer site-of-origin and 97.1% for cancer lineage. High confidence predictions (encompassing the majority of cases) were accurate 98-99% of the time in both localized and remarkably even in metastatic samples. We also identified emergent properties of our lineage scores for tumor types on which the model was never trained (zero-shot learning). Adenocarcinoma/sarcoma lineage scores differentiated epithelioid from biphasic/sarcomatoid mesothelioma. Also, predicted lineage de-differentiation identified neuroendocrine/small cell tumors and was associated with poor outcomes across tumor types. Our platform-independent single-sample approach can be easily translated to existing RNA-seq platforms. ATLAS can complement and guide traditional histopathologic assessment in challenging situations and tumors of unknown primary.
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Adenocarcinoma , Mesotelioma Maligno , Tumores Neuroendocrinos , Masculino , Humanos , Aprendizaje Automático , Adenocarcinoma/diagnóstico , Adenocarcinoma/genéticaRESUMEN
Triple negative breast cancer (TNBC) is characterized by high rates of disease recurrence after definitive therapy, and median survival of less than 18 months in the metastatic setting. Systemic therapy options for TNBC consist primarily of cytotoxic chemotherapy-containing regimens, and while recently FDA-approved chemo-immunotherapy combinations and antibody-drug conjugates such as Sacituzumab govitecan have improved clinical outcomes, there remains an unmet need for more effective and less toxic therapies. A subset of TNBC expresses the androgen receptor (AR), a nuclear hormone steroid receptor that activates an androgen-responsive transcriptional program, and gene expression profiling has revealed a TNBC molecular subtype with AR expression and luminal and androgen responsive features. Both preclinical and clinical data suggest biologic similarities between luminal AR (LAR) TNBC and ER+ luminal breast cancer, including lower proliferative activity, relative chemoresistance, and high rates of oncogenic activating mutations in the phosphatidylinositol-3-kinase (PI3K) pathway. Preclinical LAR-TNBC models are sensitive to androgen signaling inhibitors (ASIs), and particularly given the availability of FDA-approved ASIs with robust efficacy in prostate cancer, there has been great interest in targeting this pathway in AR+ TNBC. Here, we review the underlying biology and completed and ongoing androgen-targeted therapy studies in early stage and metastatic AR+ TNBC.
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Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/genética , Neoplasias de la Mama Triple Negativas/patología , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Andrógenos/farmacología , Andrógenos/uso terapéutico , Recurrencia Local de Neoplasia , Transducción de SeñalRESUMEN
BackgroundNeuroendocrine prostate cancer (NEPC) is an aggressive subtype, the presence of which changes the prognosis and management of metastatic prostate cancer.MethodsWe performed analytical validation of a Circulating Tumor Cell (CTC) multiplex RNA qPCR assay to identify the limit of quantification (LOQ) in cell lines, synthetic cDNA, and patient samples. We next profiled 116 longitudinal samples from a prospectively collected institutional cohort of 17 patients with metastatic prostate cancer (7 NEPC, 10 adenocarcinoma) as well as 265 samples from 139 patients enrolled in 3 adenocarcinoma phase II trials of androgen receptor signaling inhibitors (ARSIs). We assessed a NEPC liquid biomarker via the presence of neuroendocrine markers and the absence of androgen receptor (AR) target genes.ResultsUsing the analytical validation LOQ, liquid biomarker NEPC detection in the longitudinal cohort had a per-sample sensitivity of 51.35% and a specificity of 91.14%. However, when we incorporated the serial information from multiple liquid biopsies per patient, a unique aspect of this study, the per-patient predictions were 100% accurate, with a receiver-operating-curve (ROC) AUC of 1. In the adenocarcinoma ARSI trials, the presence of neuroendocrine markers, even while AR target gene expression was retained, was a strong negative prognostic factor.ConclusionOur analytically validated CTC biomarker can detect NEPC with high diagnostic accuracy when leveraging serial samples that are only feasible using liquid biopsies. Patients with expression of NE genes while retaining AR-target gene expression may indicate the transition to neuroendocrine differentiation, with clinical characteristics consistent with this phenotype.FundingNIH (DP2 OD030734, 1UH2CA260389, R01CA247479, and P30 CA014520), Department of Defense (PC190039 and PC200334), and Prostate Cancer Foundation (Movember Foundation - PCF Challenge Award).
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Adenocarcinoma , Neoplasias de la Próstata , Humanos , Masculino , Receptores Androgénicos/genética , Receptores Androgénicos/metabolismo , Neoplasias de la Próstata/diagnóstico , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Adenocarcinoma/patología , Biomarcadores , Transducción de Señal , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
DNA mutations in specific genes can confer preferential benefit from drugs targeting those genes. However, other molecular perturbations can "phenocopy" pathogenic mutations, but would not be identified using standard clinical sequencing, leading to missed opportunities for other patients to benefit from targeted treatments. We hypothesized that RNA phenocopy signatures of key cancer driver gene mutations could improve our ability to predict response to targeted therapies, despite not being directly trained on drug response. To test this, we built gene expression signatures in tissue samples for specific mutations and found that phenocopy signatures broadly increased accuracy of drug response predictions in-vitro compared to DNA mutation alone, and identified additional cancer cell lines that respond well with a positive/negative predictive value on par or better than DNA mutations. We further validated our results across four clinical cohorts. Our results suggest that routine RNA sequencing of tumors to identify phenocopies in addition to standard targeted DNA sequencing would improve our ability to accurately select patients for targeted therapies in the clinic.
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BACKGROUND: Expert knowledge is often shared among multidisciplinary academic teams at tumor boards (TBs) across the country, but these conversations exist in silos and do not reach the wider oncology community. OBJECTIVE: Using an oncologist-only question and answer (Q&A) website, we sought to document expert insights from TBs at National Cancer Institute-designated Comprehensive Cancer Centers (NCI-CCCs) to provide educational benefits to the oncology community. METHODS: We designed a process with the NCI-CCCs to document and share discussions from the TBs focused on areas of practice variation on theMednet, an interactive Q&A website of over 13,000 US oncologists. The faculty translated the TB discussions into concise, non-case-based Q&As on theMednet. Answers were peer reviewed and disseminated in email newsletters to registered oncologists. Reach and engagement were measured. Following each Q&A, a survey question asked how the TB Q&As impacted the readers' practice. RESULTS: A total of 23 breast, thoracic, gastrointestinal, and genitourinary programs from 16 NCI-CCC sites participated. Between December 2016 and July 2021, the faculty highlighted 368 questions from their TBs. Q&As were viewed 147,661 times by 7381 oncologists at 3515 institutions from all 50 states. A total of 277 (75%) Q&As were viewed every month. Of the 1063 responses to a survey question on how the Q&A affected clinicians' practices, 646 (61%) reported that it confirmed their current practice, 163 (20%) indicated that a Q&A would change their future practice, and 214 (15%) reported learning something new. CONCLUSIONS: Through an online Q&A platform, academics at the NCI-CCCs share knowledge outside the walls of academia with oncologists across the United States. Access to up-to-date expert knowledge can reassure clinicians' practices, significantly impact patient care in community practices, and be a source of new knowledge and education.
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PURPOSE: Liquid biopsies in metastatic renal cell carcinoma (mRCC) provide a unique approach to understand the molecular basis of treatment response and resistance. This is particularly important in the context of immunotherapies, which target key immune-tumor interactions. Unlike metastatic tissue biopsies, serial real-time profiling of mRCC is feasible with our noninvasive circulating tumor cell (CTC) approach. METHODS: We collected 457 longitudinal liquid biopsies from 104 patients with mRCC enrolled in one of two studies, either a prospective cohort or a phase II multicenter adaptive immunotherapy trial. Using a novel CTC capture and automated microscopy platform, we profiled CTC enumeration and expression of HLA I and programmed cell death-ligand 1 (PD-L1). Given their diametric immunological roles, we focused on the HLA I to PD-L1 ratio (HP ratio). RESULTS: Patients with radiographic responses showed significantly lower CTC abundances throughout treatment. Furthermore, patients whose CTC enumeration trajectory was in the highest quartile (> 0.12 CTCs/mL annually) had shorter overall survival (median 17.0 months v 21.1 months, P < .001). Throughout treatment, the HP ratio decreased in patients receiving immunotherapy but not in patients receiving tyrosine kinase inhibitors. Patients with an HP ratio trajectory in the highest quartile (≥ 0.0012 annually) displayed significantly shorter overall survival (median 18.4 months v 21.2 months, P = .003). CONCLUSION: In the first large longitudinal CTC study in mRCC to date to our knowledge, we identified the prognostic importance of CTC enumeration and the change over time of both CTC enumeration and the HP ratio. These insights into changes in both tumor burden and the molecular profile of tumor cells in response to different treatments provide potential biomarkers to predict and monitor response to immunotherapy in mRCC.
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Carcinoma de Células Renales , Neoplasias Renales , Células Neoplásicas Circulantes , Humanos , Células Neoplásicas Circulantes/patología , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/terapia , Antígeno B7-H1/metabolismo , Estudios Prospectivos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/terapia , PronósticoRESUMEN
BACKGROUND: Inhibitors of poly(ADP-ribose) polymerase (PARP) proteins potentiate antitumor activity of platinum chemotherapy. This study sought to determine the safety and tolerability of PARP inhibitor talazoparib with carboplatin and paclitaxel. METHODS: We conducted a phase I study of talazoparib with carboplatin AUC5-6 and paclitaxel 80 mg/m2 days 1, 8, 15 of 21-day cycles in patients with advanced solid tumors. Patients enrolled using a 3 + 3 design in two cohorts with talazoparib for 7 (schedule A) or 3 days (schedule B). After induction with 4-6 cycles of triplet therapy, patients received one of three maintenance options: (a) continuation of triplet (b) carboplatin/talazoparib, or (c) talazoparib monotherapy. RESULTS: Forty-three patients were treated. The MTD for both schedules was talazoparib 250mcg daily. The main toxicity was myelosuppression including grade 3/4 hematologic treatment-related adverse events (TRAEs). Dose modification occurred in 87% and 100% of patients for schedules A and B, respectively. Discontinuation due to TRAEs was 13% in schedule A and 10% in B. Ten out of 22 evaluable patients in schedule A and 5/16 patients in schedule B had a complete or partial response. Twelve out of 43 patients received ≥6 cycles of talazoparib after induction, with a 13-month median duration of maintenance. CONCLUSION: We have established the recommended phase II dose of Talazoparib at 250mcg on a 3- or 7-day schedule with carboplatin AUC6 and paclitaxel 80 mg/m2 on days 1, 8, 15 of 21-day cycles. This regimen is associated with significant myelosuppression, and in addition to maximizing supportive care, modification of the chemotherapy component would be a consideration for further development of this combination with the schedules investigated in this study.
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Neoplasias , Paclitaxel , Humanos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carboplatino , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Poli(ADP-Ribosa) PolimerasasRESUMEN
The management of early-stage human epidermal growth factor receptor 2-positive (HER2+) breast cancer has evolved in recent years, with the current standard being to tailor the intensity of adjuvant treatment to individual risk. Risk-adapted approaches to systemic therapy have been facilitated both by the recent introduction of multiple novel HER2-targeted therapies and by the development of clinical and pathologic surrogates to enable better prediction of disease behavior. These approaches have been successful at both ends of the disease spectrum. Patients with low-risk tumors now experience excellent long-term outcomes and reduced toxicity after de-escalated adjuvant therapy, and patients with high-risk residual disease after neoadjuvant systemic therapy have had a significant decrease in the risk for disease recurrence with the escalation of adjuvant therapy, including the use of trastuzumab emtansine (also known as T-DM1). We review here key developments in neoadjuvant and adjuvant systemic therapy for early-stage HER2+ breast cancer and provide an overview of the current standards of management, incorporating recent advances in escalation and de-escalation approaches for higher- and lower-risk disease, respectively. We also discuss areas of ongoing clinical uncertainty, including how disease heterogeneity and hormone receptor status affect the selection of treatments and the selection of patients for chemotherapy-free approaches. Finally, we review ongoing areas of active investigation and unmet clinical needs in this patient population.
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Neoplasias de la Mama/tratamiento farmacológico , Quimioterapia Adyuvante , Receptor ErbB-2 , Trastuzumab/uso terapéutico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Estadificación de Neoplasias , Factores de RiesgoRESUMEN
OPINION STATEMENT: Hormone receptor (HR) positive breast cancer has a high propensity for late recurrences that might be prevented with longer durations of endocrine therapy (ET). However, trials evaluating extended adjuvant ET have produced somewhat conflicting results. Additionally, ET is associated with not only day to day side effects that can impact quality of life, but more detrimental effects that can cause significant morbidity. Although patients with higher stage disease are at greater risk of late recurrences, even patients with stage 1 disease have a significant risk of recurrence after 5 years. Current guidelines recommend extending therapy for patients with node-positive disease, but recommendations for patients with node-negative disease are less clear. This has led to the development of various genomic tests to aid oncologists in further individualizing their approach when it comes to deciding which subpopulation of patients with HR-positive breast cancer may benefit from extending their endocrine therapy beyond 5 years. There are several assays that are prognostic of recurrences years 6 to 10 following diagnosis. Additionally, the breast cancer index has been shown to be predictive of extended ET in patients who have completed 5 years of tamoxifen. None of the available assays are, to date, predictive of recurrence after 10 years. Genomic testing is not appropriate for all patients, especially if the results will not predict the choice of further treatment. Ultimately, genomic testing should help facilitate shared decision making between the patient and oncologist.
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Antineoplásicos Hormonales/normas , Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Quimioterapia Adyuvante/normas , Recurrencia Local de Neoplasia/prevención & control , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Perfilación de la Expresión Génica , Humanos , Recurrencia Local de Neoplasia/genética , Recurrencia Local de Neoplasia/metabolismo , Recurrencia Local de Neoplasia/patología , PronósticoRESUMEN
Endocrine therapy has been the standard of care for patients with metastatic hormone receptor (HR)-positive, HER2-negative breast cancer since the 1970s, improving survival while avoiding the toxicities associated with cytotoxic chemotherapy. However, all HR-positive tumors ultimately develop resistance to endocrine therapy. Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors have more recently become an important component of the management of this breast cancer subtype, significantly delaying time to the disease progression and improving survival when combined with endocrine therapy. However, as with endocrine therapy alone, treatment resistance remains a universal phenomenon. As more women receive CDK4/6 inhibitors as part of their treatment, the management of de novo and acquired resistance to combined CDK4/CDK6 inhibitor plus endocrine therapy regimens has emerged as an important clinical challenge. Several resistance mechanisms have been described, including alterations in the CDK4/6/cyclin D complex or its major effector retinoblastoma protein (pRb), bypass signaling through other cyclin/CDK complexes and activation of upstream signaling pathways, in particular the PI3K/mTOR pathway, but robust biomarkers to predict resistance remain elusive, and the role for continuing CDK4/6 inhibitors after progression remains under investigation. Novel strategies being evaluated in clinical trials include the continuation of CDK4/6 inhibitors through progression, as well as triplet therapy combinations with PI3K inhibitors or immune checkpoint inhibitors.
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Neoplasias de la Mama/tratamiento farmacológico , Inhibidor p18 de las Quinasas Dependientes de la Ciclina/genética , Resistencia a Antineoplásicos/genética , Inhibidores de Proteínas Quinasas/uso terapéutico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Femenino , Humanos , Metástasis de la Neoplasia , Receptores de Estrógenos/antagonistas & inhibidores , Receptores de Estrógenos/genéticaRESUMEN
Macro-autophagy is an ancient and highly conserved self-degradative process that plays a homeostatic role in normal cells by eliminating organelles, pathogens, and protein aggregates. Autophagy, as it is routinely referred to, also allows cells to maintain metabolic sufficiency and survive under conditions of nutrient stress by recycling the by-products of autophagic degradation, such as fatty acids, amino acids, and nucleotides. Tumor cells are more reliant than normal cells on autophagy for survival in part due to their rapid growth rate, altered metabolism, and nutrient-deprived growth environment. How this dependence of tumor cells on autophagy affects their progression to malignancy and metastatic disease is an area of increasing research focus. Here, we review recent work identifying critical functions for autophagy in tumor cell migration and invasion, tumor stem cell maintenance and therapy resistance, and cross-talk between tumor cells and their microenvironment.
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Autofagia/fisiología , Metástasis de la Neoplasia , Neoplasias/patología , Microambiente Tumoral , Humanos , Invasividad Neoplásica , Células Madre Neoplásicas/patologíaRESUMEN
Autophagy inhibition is being evaluated as a novel therapeutic strategy in multiple tumor types, but little is known about its implications for metastatic dissemination. We recently reported that autophagic degradation of paxillin through direct interaction with the autophagy protein LC3B is required for focal adhesion disassembly, Src-stimulated tumor cell motility, and metastasis.
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Metastasis requires tumor cells to overcome a series of challenges to successfully travel to and colonize new microenvironments. As an adaptive (or maladaptive) response to stress, macroautophagy/autophagy has garnered increasing interest with respect to cancer metastasis, supported by clinical observations of increased autophagic flux in distant metastases relative to primary tumors. Recently, we identified a new role for autophagy in tumor cell motility through the turnover of focal adhesions, large multi-protein structures that link extracellular matrix-bound integrins to the cytoskeleton. The disassembly of focal adhesions at the cell rear is critical to forward movement and successful migration/invasion. We demonstrated that the focal adhesion protein PXN (paxillin), which serves as a crucial scaffolding and signal integrator, binds directly to LC3B through a conserved LC3-interacting region (LIR) motif to stimulate focal adhesion disassembly and metastasis and that this interaction is further promoted by oncogenic SRC.
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Autofagia , Movimiento Celular/fisiología , Adhesiones Focales/metabolismo , Neoplasias/patología , Secuencias de Aminoácidos , Adhesión Celular , Proteínas del Citoesqueleto/metabolismo , Citoesqueleto/metabolismo , Humanos , Metástasis de la Neoplasia , Neoplasias/metabolismo , Paxillin/metabolismo , Fosforilación , Transducción de Señal/fisiologíaRESUMEN
Autophagy is a conserved catabolic process that plays a housekeeping role in eliminating protein aggregates and organelles and is activated during nutrient deprivation to generate metabolites and energy. Autophagy plays a significant role in tumorigenesis, although opposing context-dependent functions of autophagy in cancer have complicated efforts to target autophagy for therapeutic purposes. We demonstrate that autophagy inhibition reduces tumor cell migration and invasion in vitro and attenuates metastasis in vivo. Numerous abnormally large focal adhesions (FAs) accumulate in autophagy-deficient tumor cells, reflecting a role for autophagy in FA disassembly through targeted degradation of paxillin. We demonstrate that paxillin interacts with processed LC3 through a conserved LIR motif in the amino-terminal end of paxillin and that this interaction is regulated by oncogenic SRC activity. Together, these data establish a function for autophagy in FA turnover, tumor cell motility, and metastasis.
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Autofagia , Movimiento Celular , Adhesiones Focales/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neoplasias/patología , Paxillin/metabolismo , Familia-src Quinasas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Autofagosomas/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Línea Celular Tumoral , Proliferación Celular , Técnicas de Silenciamiento del Gen , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Paxillin/química , Unión Proteica , Estabilidad Proteica , Transporte de ProteínasRESUMEN
The oligomeric Mg chelatase (MgCh), consisting of the subunits CHLH, CHLI, and CHLD, is located at the central site of chlorophyll synthesis, but is also thought to have an additional function in regulatory feedback control of the tetrapyrrole biosynthesis pathway and in chloroplast retrograde signaling. In Arabidopsis thaliana and Chlamydomonas reinhardtii, two genes have been proposed to encode the CHLI subunit of MgCh. While the role of CHLI1 in A. thaliana MgCh has been substantially elucidated, different reports provide inconsistent results with regard to the function of CHLI2 in Mg chelation and retrograde signaling. In the present report, the possible functions of both isoforms were analyzed in C. reinhardtii Knockout of the CHLI1 gene resulted in complete loss of MgCh activity, absence of chlorophyll, acute light sensitivity, and, as a consequence, down-regulation of tetrapyrrole biosynthesis and photosynthesis-associated nuclear genes. These observations indicate a phenotypical resemblance of chli1 to the chlh and chld C. reinhardtii mutants previously reported. The key role of CHLI1 for MgCh reaction in comparison with the second isoform was confirmed by the rescue of chli1 with genomic CHLI1 Because CHLI2 in C. reinhardtii shows lower expression than CHLI1, strains overexpressing CHLI2 were produced in the chli1 background. However, no complementation of the chli1 phenotype was observed. Silencing of CHLI2 in the wild-type background did not result in any changes in the accumulation of tetrapyrrole intermediates or of chlorophyll. The results suggest that, unlike in A. thaliana, changes in CHLI2 content observed in the present studies do not affect formation and activity of MgCh in C. reinhardtii.
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Proteínas Algáceas/genética , Chlamydomonas reinhardtii/genética , Liasas/genética , Transducción de Señal , Proteínas Algáceas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Clorofila/metabolismo , Regulación hacia Abajo , Liasas/metabolismo , Filogenia , Análisis de Secuencia de ADN , Tetrapirroles/metabolismoRESUMEN
BNip3 is a hypoxia-inducible protein that targets mitochondria for autophagosomal degradation. We report a novel tumor suppressor role for BNip3 in a clinically relevant mouse model of mammary tumorigenesis. BNip3 delays primary mammary tumor growth and progression by preventing the accumulation of dysfunctional mitochondria and resultant excess ROS production. In the absence of BNip3, mammary tumor cells are unable to reduce mitochondrial mass effectively and elevated mitochondrial ROS increases the expression of Hif-1α and Hif target genes, including those involved in glycolysis and angiogenesistwo processes that are also markedly increased in BNip3-null tumors. Glycolysis inhibition attenuates the growth of BNip3-null tumor cells, revealing an increased dependence on autophagy for survival. We also demonstrate that BNIP3 deletion can be used as a prognostic marker of tumor progression to metastasis in human triple-negative breast cancer (TNBC). These studies show that mitochondrial dysfunctioncaused by defects in mitophagycan promote the Warburg effect and tumor progression, and suggest better approaches to stratifying TNBC for treatment.
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Neoplasias Mamarias Experimentales/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mitofagia , Neoplasias de la Mama Triple Negativas/patología , Animales , Biomarcadores de Tumor/análisis , Progresión de la Enfermedad , Femenino , Glucólisis , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/secundario , Neoplasias Mamarias Experimentales/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Proteínas Mitocondriales/deficiencia , Metástasis de la Neoplasia , Neovascularización Patológica/metabolismo , Pronóstico , Especies Reactivas de Oxígeno/metabolismo , Neoplasias de la Mama Triple Negativas/metabolismoRESUMEN
Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5-Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.
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Autofagia/fisiología , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteína 5 Relacionada con la Autofagia , Western Blotting , Humanos , Inmunohistoquímica , Macrólidos/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Sequestosoma-1RESUMEN
Chlamydomonas reinhardtii is a unicellular green alga that is a key model organism in the study of photosynthesis and oxidative stress. Here we describe the large-scale generation of a population of insertional mutants that have been screened for phenotypes related to photosynthesis and the isolation of 459 flanking sequence tags from 439 mutants. Recent phylogenomic analysis has identified a core set of genes, named GreenCut2, that are conserved in green algae and plants. Many of these genes are likely to be central to the process of photosynthesis, and they are over-represented by sixfold among the screened insertional mutants, with insertion events isolated in or adjacent to 68 of 597 GreenCut2 genes. This enrichment thus provides experimental support for functional assignments based on previous bioinformatic analysis. To illustrate one of the uses of the population, a candidate gene approach based on genome position of the flanking sequence of the insertional mutant CAL027_01_20 was used to identify the molecular basis of the classical C. reinhardtii mutation ac17. These mutations were shown to affect the gene PDH2, which encodes a subunit of the plastid pyruvate dehydrogenase complex. The mutants and associated flanking sequence data described here are publicly available to the research community, and they represent one of the largest phenotyped collections of algal insertional mutants to date.
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Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Fotosíntesis/genética , Proteínas de Plantas/metabolismo , Acetatos/metabolismo , Mutagénesis Insercional , Mutación , Proteínas de Plantas/genéticaRESUMEN
The phosphoinositol-3 kinase (PI3K) pathway is highly dysregulated in squamous cell carcinoma of the head and neck (SCCHN). While inhibitors of the PI3K/AKT pathway are being developed in cancer, their efficacy does not appear to be related to the presence of mutations or amplification in pathway genes. The PI3K pathway is a major regulator of macro-autophagy, an evolutionarily conserved catabolic process that degrades cellular materials to promote cellular homeostasis and survival under stress. Employing a panel of SCCHN cell lines, we observed a significant correlation between the activity of PI3K/AKT inhibitors and their ability to induce autophagy. More specifically, resistance to these inhibitors was associated with accumulation of p62/SQSTM1, a pleotropic protein that is consumed during autophagy, while loss of autophagy was, for the first time, found to be due to silencing of an essential autophagy gene, ATG7. Moreover, modulating ATG7 and p62/SQSTM1 could regulate sensitivity to PI3K/AKT inhibitors, underscoring a mechanistic link between autophagy and drug sensitivity. Analysis of human tissues revealed progressive accumulation of p62/SQSTM1 in a significant proportion of cancer samples compared to normal tissue, suggesting that defective autophagy has relevance to SCCHN. These findings are further validated by analysis of TCGA data confirming homozygous deletion and mRNA down-regulation of ATG7 in 10.0% of SCCHN samples. Taken together, these data indicate that p62/SQSTM1 levels modulate sensitivity to PI3K/AKT inhibitors; cancers vary in their capacity to undergo autophagy through epigenetic modification and, when deficient, accumulate p62/SQSTM1; and expression of autophagy-related proteins may serve as markers for resistance to PI3K/AKT inhibitors in SCCHN.