ABSTRACT
Ubiquitination is one of the most common posttranslational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity toward K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage-dependent manner, thus serving as an interpreter of the ubiquitin code.
Subject(s)
Adaptor Proteins, Signal Transducing , Autophagy-Related Proteins , Ubiquitination , Autophagy-Related Proteins/metabolism , Autophagy-Related Proteins/chemistry , Autophagy-Related Proteins/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Humans , Polyubiquitin/metabolism , Proteasome Endopeptidase Complex/metabolism , Biomolecular Condensates/metabolism , Biomolecular Condensates/chemistry , Ubiquitin/metabolism , Ubiquitin/chemistry , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/chemistry , Cell Cycle Proteins/genetics , Ubiquitinated Proteins/metabolism , Ubiquitinated Proteins/isolation & purification , Ubiquitinated Proteins/chemistry , Phase SeparationABSTRACT
BACKGROUND: The influence of Helicobacter-pylori (H. pylori) infection and the characteristics of gastric cancer (GC) on tumor-infiltrating lymphocyte (TIL) levels has not been extensively studied. Analysis of infiltrating-immune-cell subtypes as well as survival is necessary to obtain comprehensive information. AIM: To determine the rates of deficient mismatch-repair (dMMR), HER2-status and H. pylori infection and their association with TIL levels in GC. METHODS: Samples from 503 resected GC tumors were included and TIL levels were evaluated following the international-TILs-working-group recommendations with assessment of the intratumoral (IT), stromal (ST) and invasive-border (IB) compartments. The density of CD3, CD8 and CD163 immune cells, and dMMR and HER2-status were determined by immunohistochemistry (IHC). H. pylori infection was evaluated by routine histology and quantitative PCR (qPCR) in a subset of samples. RESULTS: dMMR was found in 34.4%, HER2+ in 5% and H. pylori-positive in 55.7% of samples. High IT-TIL was associated with grade-3 (P = 0.038), while ST-TIL with grade-1 (P < 0.001), intestinal-histology (P < 0.001) and no-recurrence (P = 0.003). dMMR was associated with high TIL levels in the ST (P = 0.019) and IB (P = 0.01) compartments, and ST-CD3 (P = 0.049) and ST-CD8 (P = 0.05) densities. HER2- was associated with high IT-CD8 (P = 0.009). H. pylori-negative was associated with high IT-TIL levels (P = 0.009) when assessed by routine-histology, and with high TIL levels in the 3 compartments (P = 0.002-0.047) and CD8 density in the IT and ST compartments (P = 0.001) when assessed by qPCR. A longer overall survival was associated with low IT-CD163 (P = 0.003) and CD8/CD3 (P = 0.001 in IT and P = 0.002 in ST) and high IT-CD3 (P = 0.021), ST-CD3 (P = 0.003) and CD3/CD163 (P = 0.002). CONCLUSION: TIL levels were related to dMMR and H. pylori-negativity. Low CD8/CD3 and high CD163/CD3 were associated with lower recurrence and longer survival.
ABSTRACT
The cytoskeleton is a complex network of interconnected biopolymers consisting of actin filaments, microtubules, and intermediate filaments. These biopolymers work in concert to transmit cell-generated forces to the extracellular matrix required for cell motility, wound healing, and tissue maintenance. While we know cell-generated forces are driven by actomyosin contractility and balanced by microtubule network resistance, the effect of intermediate filaments on cellular forces is unclear. Using a combination of theoretical modeling and experiments, we show that vimentin intermediate filaments tune cell stress by assisting in both actomyosin-based force transmission and reinforcement of microtubule networks under compression. We show that the competition between these two opposing effects of vimentin is regulated by the microenvironment stiffness. These results reconcile seemingly contradictory results in the literature and provide a unified description of vimentin's effects on the transmission of cell contractile forces to the extracellular matrix.
Subject(s)
Actomyosin , Mechanotransduction, Cellular , Microtubules , Vimentin , Microtubules/metabolism , Actomyosin/metabolism , Vimentin/metabolism , Humans , Extracellular Matrix/metabolism , AnimalsABSTRACT
Ubiquitination is one of the most common post-translational modifications in eukaryotic cells. Depending on the architecture of polyubiquitin chains, substrate proteins can meet different cellular fates, but our understanding of how chain linkage controls protein fate remains limited. UBL-UBA shuttle proteins, such as UBQLN2, bind to ubiquitinated proteins and to the proteasome or other protein quality control machinery elements and play a role in substrate fate determination. Under physiological conditions, UBQLN2 forms biomolecular condensates through phase separation, a physicochemical phenomenon in which multivalent interactions drive the formation of a macromolecule-rich dense phase. Ubiquitin and polyubiquitin chains modulate UBQLN2's phase separation in a linkage-dependent manner, suggesting a possible link to substrate fate determination, but polyubiquitinated substrates have not been examined directly. Using sedimentation assays and microscopy we show that polyubiquitinated substrates induce UBQLN2 phase separation and incorporate into the resulting condensates. This substrate effect is strongest with K63-linked substrates, intermediate with mixed-linkage substrates, and weakest with K48-linked substrates. Proteasomes can be recruited to these condensates, but proteasome activity towards K63-linked and mixed linkage substrates is inhibited in condensates. Substrates are also protected from deubiquitinases by UBQLN2-induced phase separation. Our results suggest that phase separation could regulate the fate of ubiquitinated substrates in a chain-linkage dependent manner, thus serving as an interpreter of the ubiquitin code.
ABSTRACT
The ubiquitin-adaptor protein UBQLN2 promotes degradation of several aggregate-prone proteins implicated in neurodegenerative diseases. Missense UBQLN2 mutations also cause X-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previously we demonstrated that the liquid-like properties of UBQLN2 molecular assemblies are altered by a specific pathogenic mutation, P506T, and that the propensity of UBQLN2 to aggregate correlated with neurotoxicity. Here, we systematically assess the effects of multiple, spatially distinct ALS/FTD-linked missense mutations on UBQLN2 aggregation propensity, neurotoxicity, phase separation, and autophagic flux. In contrast to what we observed for the P506T mutation, no other tested pathogenic mutant exhibited a clear correlation between aggregation propensity and neurotoxicity. These results emphasize the unique nature of pathogenic UBQLN2 mutations and argue against a generalizable link between aggregation propensity and neurodegeneration in UBQLN2-linked ALS/FTD.
Subject(s)
Amyotrophic Lateral Sclerosis , Frontotemporal Dementia , Humans , Frontotemporal Dementia/genetics , Amyotrophic Lateral Sclerosis/metabolism , Autophagy-Related Proteins/genetics , Mutation , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2, and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates, and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2, and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2, and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs and the need to apply caution when using epitope tags to prevent experimental artifacts.
ABSTRACT
Highly homologous ubiquitin-binding shuttle proteins UBQLN1, UBQLN2 and UBQLN4 differ in both their specific protein quality control functions and their propensities to localize to stress-induced condensates, cellular aggregates and aggresomes. We previously showed that UBQLN2 phase separates in vitro, and that the phase separation propensities of UBQLN2 deletion constructs correlate with their ability to form condensates in cells. Here, we demonstrated that full-length UBQLN1, UBQLN2 and UBQLN4 exhibit distinct phase behaviors in vitro. Strikingly, UBQLN4 phase separates at a much lower saturation concentration than UBQLN1. However, neither UBQLN1 nor UBQLN4 phase separates with a strong temperature dependence, unlike UBQLN2. We determined that the temperature-dependent phase behavior of UBQLN2 stems from its unique proline-rich (Pxx) region, which is absent in the other UBQLNs. We found that the short N-terminal disordered regions of UBQLN1, UBQLN2 and UBQLN4 inhibit UBQLN phase separation via electrostatics interactions. Charge variants of the N-terminal regions exhibit altered phase behaviors. Consistent with the sensitivity of UBQLN phase separation to the composition of the N-terminal regions, epitope tags placed on the N-termini of the UBQLNs tune phase separation. Overall, our in vitro results have important implications for studies of UBQLNs in cells, including the identification of phase separation as a potential mechanism to distinguish the cellular roles of UBQLNs, and the need to apply caution when using epitope tags to prevent experimental artifacts.
ABSTRACT
Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or posttranslational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to either the UBQLN2-binding surface of Ub or the spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model based on polyphasic linkage principles that accurately described the effects of different hubs on UBQLN2 phase separation, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. The extent to which polyubiquitin hubs promote UBQLN2 phase separation is encoded in the spacings between Ub units. This spacing is modulated by chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. The spacing in naturally occurring linear polyubiquitin chains is already optimized to promote phase separation with UBQLN2. We expect our findings to extend to other condensates, emphasizing the importance of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
Subject(s)
Polyubiquitin , Ubiquitin , Ubiquitin/metabolism , Polyubiquitin/metabolism , Ligands , Ubiquitination , Binding SitesABSTRACT
Liquid-liquid phase separation (LLPS) is a process by which biomacromolecules, particularly proteins, condense into a dense phase that resembles liquid droplets. Dysregulation of LLPS is implicated in disease, yet the relationship between protein conformational changes and LLPS remains difficult to discern. This is due to the high flexibility and disordered nature of many proteins that phase separate under physiological conditions and their tendency to oligomerize. Here, we demonstrate that ion mobility mass spectrometry (IM-MS) overcomes these limitations. We used IM-MS to investigate the conformational states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which is driven by high-salt concentration and reversed by noncovalent interactions with ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as a mixture of monomers and dimers and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift toward extended conformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes, which have compact geometries compared to free UBQLN2 dimers. Together, these results suggest that extended conformations of UBQLN2 are correlated with UBQLN2's ability to phase separate. Overall, delineating protein conformations that are implicit in LLPS will greatly increase understanding of the phase separation process, both in normal cell physiology and disease states.
Subject(s)
Transcription Factors , Ubiquitin , Protein Conformation , Mass SpectrometryABSTRACT
In this issue of Structure, Buel et al. (2023) combined NMR data with AlphaFold2 to map out the interaction between the AZUL domain of ubiquitin ligase E6AP and UBQLN1/2 UBA. The authors demonstrated that this interaction enhances the self-association of the helix neighboring UBA and enables E6AP to localize to UBQLN2 droplets.
Subject(s)
Ubiquitin-Protein Ligases , Ubiquitin , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/chemistry , Protein BindingABSTRACT
Introduction: Breast cancer is a heterogeneous disease, and the distribution of the different subtypes varies by race/ethnic category in the United States and by country. Established breast cancer-associated factors impact subtype-specific risk; however, these included limited or no representation of Latin American diversity. To address this gap in knowledge, we report a description of demographic, reproductive, and lifestyle breast cancer-associated factors by age at diagnosis and disease subtype for The Peruvian Genetics and Genomics of Breast Cancer (PEGEN-BC) study. Methods: The PEGEN-BC study is a hospital-based breast cancer cohort that includes 1943 patients diagnosed at the Instituto Nacional de Enfermedades Neoplásicas in Lima, Peru. Demographic and reproductive information, as well as lifestyle exposures, were collected with a questionnaire. Clinical data, including tumor Hormone Receptor (HR) status and Human Epidermal Growth Factor Receptor 2 (HER2) status, were abstracted from electronic medical records. Differences in proportions and mean values were tested using Chi-squared and one-way ANOVA tests, respectively. Multinomial logistic regression models were used for multivariate association analyses. Results: The distribution of subtypes was 52% HR+HER2-, 19% HR+HER2+, 16% HR-HER2-, and 13% HR-HER2+. Indigenous American (IA) genetic ancestry was higher, and height was lower among individuals with the HR-HER2+ subtype (80% IA vs. 76% overall, p=0.007; 152 cm vs. 153 cm overall, p=0.032, respectively). In multivariate models, IA ancestry was associated with HR-HER2+ subtype (OR=1.38,95%CI=1.06-1.79, p=0.017) and parous women showed increased risk for HR-HER2+ (OR=2.7,95%CI=1.5-4.8, p<0.001) and HR-HER2- tumors (OR=2.4,95%CI=1.5-4.0, p<0.001) compared to nulliparous women. Multiple patient and tumor characteristics differed by age at diagnosis (<50 vs. >=50), including ancestry, region of residence, family history, height, BMI, breastfeeding, parity, and stage at diagnosis (p<0.02 for all variables). Discussion: The characteristics of the PEGEN-BC study participants do not suggest heterogeneity by tumor subtype except for IA genetic ancestry proportion, which has been previously reported. Differences by age at diagnosis were apparent and concordant with what is known about pre- and post-menopausal-specific disease risk factors. Additional studies in Peru should be developed to further understand the main contributors to the specific age of onset and molecular disease subtypes in this population and develop population-appropriate predictive models for prevention.
ABSTRACT
Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Perturbations to the UBQLN2-binding surface of Ub or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. By developing an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty antagonizes the ability of polyUb hubs to scaffold multiple UBQLN2 molecules and cooperatively amplify phase separation. Importantly, the extent to which polyubiquitin hubs can promote UBQLN2 phase separation are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.
ABSTRACT
Liquid-liquid phase separation (LLPS) is hypothesized to be the underlying mechanism for how membraneless organelles or biomolecular condensates form inside both prokaryotic and eukaryotic cells. Protein LLPS is a biophysical process during which proteins demix from homogeneous solution to form protein-dense droplets with liquid-like properties. Disruptions to LLPS, such as changes to material properties of condensates or physicochemical parameters for LLPS onset, are implicated in neurodegenerative diseases and cancer. Therefore, it is essential to determine the physicochemical parameters that promote protein LLPS. Here, we present our UV-Vis spectrophotometric turbidity assay to characterize the temperature and concentration dependence of LLPS for UBQLN2, a protein that undergoes LLPS via homotypic interactions in vitro and forms stress-induced condensates in cells. Mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) and disrupt UBQLN2 LLPS. We present a detailed expression and purification protocol for a C-terminal construct of UBQLN2 and how we use microscopy to image UBQLN2 LLPS. We use our UV-Vis assay to construct temperature-concentration phase diagrams for wild-type and mutant UBQLN2 constructs to determine the effects of domain deletions and/or mutations on UBQLN2 phase separation.
Subject(s)
Amyotrophic Lateral Sclerosis , Biochemical Phenomena , Humans , Amyotrophic Lateral Sclerosis/genetics , Mutation , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
OBJECTIVE: To evaluate the relationship between circulating tumor DNA (ctDNA) presence and tumor features including tumor-infiltrating lymphocyte (TIL) levels in Peruvian breast cancer patients. MATERIALS AND METHODS: This was a prospective study conducted at the Instituto Nacional de Enfemedades Neoplasicas, Peru. We evaluated level of TIL and PIK3CA mutations in ctDNA. Clinical characteristics, including outcome data, were collected from the patient file. Survival was calculated from the date of blood sample drawn to the event time. Data collected were analyzed using SPSS software version 25. RESULTS: We analyzed plasma samples from 183 breast cancer patients. most cases were of Luminal-B (44.8%) phenotype and stage II (41.5%), and median stromal TIL was 30%. PIK3CA mutation in ctDNA was detected in 35% cases (most with E545K) and was associated with lower TIL level (p=0.04). PIK3CA in ctDNA tended to be associated with advanced stages (p=0.09) in the whole series and with higher recurrence rates (p=0.053) in the non-metastatic setting. Patients with presence of PIK3CA in ctDNA tended to have shorter survival (p=0.083). CONCLUSION: Presence of PIK3CA mutation in ctDNA was frequently found in our Peruvian breast cancer series, was associated with lower TIL levels and tended to predict poor outcomes.
Subject(s)
Circulating Tumor DNA , Neoplasms , Lymphocytes, Tumor-Infiltrating/pathology , Peru , Prospective Studies , Class I Phosphatidylinositol 3-Kinases/genetics , Circulating Tumor DNA/genetics , Mutation , Biomarkers, Tumor/genetics , Neoplasms/pathologyABSTRACT
BACKGROUND: Breast cancer incidence in the United States is lower in Hispanic/Latina (H/L) compared with African American/Black or Non-Hispanic White women. An Indigenous American breast cancer-protective germline variant (rs140068132) has been reported near the estrogen receptor 1 gene. This study tests the association of rs140068132 and other polymorphisms in the 6q25 region with subtype-specific breast cancer risk in H/Ls of high Indigenous American ancestry. METHODS: Genotypes were obtained for 5,094 Peruvian women with (1,755) and without (3,337) breast cancer. Associations between genotype and overall and subtype-specific risk for the protective variant were tested using logistic regression models and conditional analyses, including other risk-associated polymorphisms in the region. RESULTS: We replicated the reported association between rs140068132 and breast cancer risk overall [odds ratio (OR), 0.53; 95% confidence interval (CI), 0.47-0.59], as well as the lower odds of developing hormone receptor negative (HR-) versus HR+ disease (OR, 0.77; 95% CI, 0.61-0.97). Models, including HER2, showed further heterogeneity with reduced odds for HR+HER2+ (OR, 0.68; 95% CI, 0.51-0.92), HR-HER2+ (OR, 0.63; 95% CI, 0.44-0.90) and HR-HER2- (OR, 0.77; 95% CI, 0.56-1.05) compared with HR+HER2-. Inclusion of other risk-associated variants did not change these observations. CONCLUSIONS: The rs140068132 polymorphism is associated with decreased risk of breast cancer in Peruvians and is more protective against HR- and HER2+ diseases independently of other breast cancer-associated variants in the 6q25 region. IMPACT: These results could inform functional analyses to understand the mechanism by which rs140068132-G reduces risk of breast cancer development in a subtype-specific manner. They also illustrate the importance of including diverse individuals in genetic studies.
Subject(s)
Breast Neoplasms , Breast Neoplasms/epidemiology , Breast Neoplasms/ethnology , Breast Neoplasms/genetics , Chromosomes, Human, Pair 6 , Female , Hispanic or Latino , Humans , Logistic Models , Peru/epidemiology , Receptor, ErbB-2/genetics , Receptors, Estrogen/genetics , Receptors, Progesterone/geneticsABSTRACT
Ubiquitin-binding shuttle UBQLN2 mediates crosstalk between proteasomal degradation and autophagy, likely via interactions with K48- and K63-linked polyubiquitin chains, respectively. UBQLN2 comprises self-associating regions that drive its homotypic liquid-liquid phase separation (LLPS). Specific interactions between one of these regions and ubiquitin inhibit UBQLN2 LLPS. Here, we show that, unlike ubiquitin, the effects of multivalent polyubiquitin chains on UBQLN2 LLPS are highly dependent on chain types. Specifically, K11-Ub4 and K48-Ub4 chains generally inhibit UBQLN2 LLPS, whereas K63-Ub4, M1-Ub4 chains, and a designed tetrameric ubiquitin construct significantly enhance LLPS. We demonstrate that these opposing effects stem from differences in chain conformations but not in affinities between chains and UBQLN2. Chains with extended conformations and increased accessibility to the ubiquitin-binding surface promote UBQLN2 LLPS by enabling a switch between homotypic to partially heterotypic LLPS that is driven by both UBQLN2 self-interactions and interactions between multiple UBQLN2 units with each polyubiquitin chain. Our study provides mechanistic insights into how the structural and conformational properties of polyubiquitin chains contribute to heterotypic LLPS with ubiquitin-binding shuttles and adaptors.
Subject(s)
Polyubiquitin , Ubiquitin , Models, Molecular , Polyubiquitin/metabolism , Protein Binding , Ubiquitin/metabolism , UbiquitinationABSTRACT
OBJECTIVE: To evaluate the frequency distribution of viral infections in Peruvian Breast Cancer (BC) lesions and its association with clinicopathological features. Additionally, a prospective evaluation of p16 and Tumor-infiltrating lymphocytes (TIL) levels were performed for developing a comprehensive analysis. METHODS: Detection of high risk- human papillomavirus (HR- HPV) through qPCR was performed in 447 BC and 79 non-cancer frozen samples. Paired paraffin samples from 238 BC were stained with Human cytomegalovirus (HCMV) and p16 immunohistochemistry. TIL was calculated in 397 BC cases. RESULTS: HCMV was positive in 72.5%. HR- HPV was detected in 2.9% of BC and 1.3% of non-malignant samples. P16+ was found in 28.15% and median TIL percentage was 30. HR- HPV infection was associated with non-ductal histology (p=0.003) and p16+ (p=0.017). Positive P16+ was associated with higher T stage (p=0.022), grade (p=0.009), TIL level (p=0.002), and triple-negative phenotype (p=0.021). CONCLUSION: HCMV is frequent, but HR- HPV infection is unusual in Peruvian BC. P16+ is associated with HR- PVH infection, high TIL and aggressive features.
Subject(s)
Alphapapillomavirus , Breast Neoplasms , Cytomegalovirus Infections , Papillomavirus Infections , Alphapapillomavirus/genetics , Biomarkers, Tumor/analysis , Breast Neoplasms/complications , Breast Neoplasms/epidemiology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cytomegalovirus Infections/complications , Cytomegalovirus Infections/epidemiology , Female , Humans , Papillomaviridae/genetics , Papillomavirus Infections/complications , Peru/epidemiology , Staining and LabelingABSTRACT
Triple-negative breast cancer (TNBC) is a highly complex, heterogeneous disease and historically has limited treatment options. It has a high probability of disease recurrence and rapid disease progression despite adequate systemic treatment. Immunotherapy has emerged as an important alternative in the management of this malignancy, showing an impact on progression-free survival and overall survival in selected populations. In this review we focused on immunotherapy and its current relevance in the management of TNBC, including various scenarios (metastatic and early -neoadjuvant, adjuvant-), new advances in this subtype and the research of potential predictive biomarkers of response to treatment.
ABSTRACT
BACKGROUND: Breast cancer (BC) frequency in males is extremely low and tumor features vary from its female counterpart. Breast cancer clinical and pathological features differ by race in women. Tumor infiltrating lymphocyte (TIL) levels, mismatch repair (MMR) protein loss, androgen receptor (AR) expression, and PIK3CA gene mutations are predictive biomarkers of response to biological therapy in female BC. There is limited information about clinical and pathological features as well as predictive biomarkers in males of non-Caucasian races with BC. AIM: To investigate clinicopathological features and biomarkers of BC tumors in males and their prognostic value in Peruvian population. METHODS: This study looked at a single-institution series of 54 Peruvian males with invasive BC who were diagnosed from Jan 2004 to June 2018. Standard pathological features, TIL levels, MMR proteins, AR immunohistochemistry staining, and PIK3CA gene mutations were prospectively evaluated in cases with available paraffin material. Percentage of AR and estrogen receptor (ER) positive cells was additionally calculated by software after slide scanning. Statistical analyses included association tests, intraclass correlation test and Kaplan Meier overall survival curves. RESULTS: The median age was 63 years and most cases were ER-positive (85.7%), HER2 negative (87.2%), Luminal-A phenotype (60%) and clinical stage II (41.5%) among our male breast tumors. Median TIL was 10% and higher levels tended to be associated with Luminal-B phenotype and higher grade. AR-positive was found in 85.3% and was correlated with ER (intraclass index of 0.835, P < 0.001). Loss of MMR proteins was found in 15.4% and PIK3CA mutation (H1047R) in 14.3% (belonged to the Luminal-A phenotype). Loss of MMR proteins was associated with AR-negative (P = 0.018) but not with ER (P = 0.43) or TIL (P = 0.84). Early stages (P < 0.001) and lower grade (P = 0.006) were associated with longer overall survival. ER status, phenotype, AR status, TIL level, MMR protein loss nor PIK3CA mutation was not associated with survival (P > 0.05). CONCLUSION: Male BC is usually ER and AR positive, and Luminal-A. MMR loss and PIK3CA mutations are infrequent. Stage and grade predicted overall survival in our South American country population.
ABSTRACT
Mutations in ubiquilin-2 (UBQLN2), a ubiquitin-binding shuttle protein involved in several protein quality control processes, can lead to amyotrophic lateral sclerosis (ALS). We previously found that wild-type UBQLN2 forms dynamic, membraneless biomolecular condensates upon cellular stress, and undergoes liquid-liquid phase separation in vitro. However, the impact of ALS-linked mutations on UBQLN2 condensate formation in cells remains unknown. Here, we overexpress mCherry-fused UBQLN2 with five patient-derived ALS-linked mutations and employ live-cell imaging and photokinetic analysis to investigate how each of these mutations impact stress-induced UBQLN2 condensate assembly and condensate material properties. Unlike endogenous UBQLN2, exogenously introduced UBQLN2 forms condensates distinct from stress granules. Both wild-type and mutant UBQLN2 condensates are generally cytoplasmic and liquid-like. However, mutant UBQLN2 forms fewer stress-induced UBQLN2 condensates than wild-type UBQLN2. Exogenously expressed P506T UBQLN2 forms the lowest number of stress-induced condensates of all UBQLN2 mutants, and these condensates are significantly smaller than those of wild-type UBQLN2. Fluorescence recovery after photobleaching (FRAP) analysis of UBQLN2 condensates revealed higher immobile fractions for UBQLN2 mutants, especially P506T. P497S and P497H mutations differentially impact condensate properties, demonstrating that the effects of ALS-linked mutations are both position- and amino acid-dependent. Collectively, our data show that disease mutations hinder assembly and alter viscoelastic properties of stress-induced UBQLN2 condensates, potentially leading to aggregates commonly observed in ALS.