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Despite advances in genomic classification of breast cancer, current clinical tests and treatment decisions are commonly based on protein level information. Formalin-fixed paraffin-embedded (FFPE) tissue specimens with extended clinical outcomes are widely available. Here, we perform comprehensive proteomic profiling of 300 FFPE breast cancer surgical specimens, 75 of each PAM50 subtype, from patients diagnosed in 2008-2013 (n = 178) and 1986-1992 (n = 122) with linked clinical outcomes. These two cohorts are analyzed separately, and we quantify 4214 proteins across all 300 samples. Within the aggressive PAM50-classified basal-like cases, proteomic profiling reveals two groups with one having characteristic immune hot expression features and highly favorable survival. Her2-Enriched cases separate into heterogeneous groups differing by extracellular matrix, lipid metabolism, and immune-response features. Within 88 triple-negative breast cancers, four proteomic clusters display features of basal-immune hot, basal-immune cold, mesenchymal, and luminal with disparate survival outcomes. Our proteomic analysis characterizes the heterogeneity of breast cancer in a clinically-applicable manner, identifies potential biomarkers and therapeutic targets, and provides a resource for clinical breast cancer classification. Protein level information enables the identification of potential biomarkers and therapeutic targets for breast cancer. Here, the authors perform proteomic analysis of 2 cohorts of breast cancer surgical specimens and identify distinct subtypes, immune features and survival outcomes.

Breast cancer heterogeneity has made it challenging to identify mechanisms critical to the initial stages of their genesis in vivo. Here, we sought to interrogate the role of YB-1 in newly arising human breast cancers as well as in established cell lines. In a first series of experiments, we found that short-hairpin RNA-mediated knockdown of YB-1 in MDA-MB-231 cells blocked both their local tumour-forming and lung-colonising activity in immunodeficient mice. Conversely, upregulated expression of YB-1 enhanced the poor in vivo tumorigenicity of T47D cells. We then found that YB-1 knockdown also inhibits the initial generation in mice of invasive ductal carcinomas and ductal carcinomas in situ from freshly isolated human mammary cells transduced, respectively, with KRASG12D or myristoylated-AKT1. Interestingly, increased expression of HIF1α and G3BP1, two YB-1 translational targets and elements of a stress-adaptive programme, mirrored the levels of YB-1 in both transformed primary and established MDA-MB-231 breast cancer cells.

Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT) is a rare and aggressive form of ovarian cancer. SCCOHT tumors have inactivating mutations in SMARCA4 (BRG1), one of the two mutually exclusive ATPases of the SWI/SNF chromatin remodeling complex. To address the role that BRG1 loss plays in SCCOHT tumorigenesis, we performed integrative multi-omic analyses in SCCOHT cell lines +/- BRG1 reexpression. BRG1 reexpression induced a gene and protein signature similar to an epithelial cell and gained chromatin accessibility sites correlated with other epithelial originating TCGA tumors. Gained chromatin accessibility and BRG1 recruited sites were strongly enriched for transcription-factor-binding motifs of AP-1 family members. Furthermore, AP-1 motifs were enriched at the promoters of highly upregulated epithelial genes. Using a dominant-negative AP-1 cell line, we found that both AP-1 DNA-binding activity and BRG1 reexpression are necessary for the gene and protein expression of epithelial genes. Our study demonstrates that BRG1 reexpression drives an epithelial-like gene and protein signature in SCCOHT cells that depends upon by AP-1 activity.

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequencing data from 1,727 unique individual patients with AML, which revealed mutations in ubiquitin ligase family genes in 11.2% of samples from adult patients with AML with mutual exclusivity. The SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex gene, FBXO11, was the most significantly downregulated gene of the SCF complex in AML. We found that FBXO11 interacts with and catalyzes K63-linked ubiquitination of LONP1 in the cytosol, to promote LONP1 entry into mitochondria. We show that depletion of FBXO11 or LONP1 reduced mitochondrial respiration through impaired LONP1 chaperone activity to assemble electron transport chain Complex IV. Reduced mitochondrial respiration secondary to FBXO11 or LONP1 depletion imparted myeloid-biased stem cell properties in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro. In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRASG12D to generate serially transplantable AML. Our findings suggest that reduced FBXO11 cooperates to initiate AML by priming HSPC for myeloid-biased self renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.

The t(X,17) chromosomal translocation, generating the ASPSCR1::TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCCs), frustrating efforts to identify therapeutic targets for these rare cancers. Here, proteomic analysis identifies VCP/p97, an AAA+ ATPase with known segregase function, as strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1::TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1::TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributes with ASPSCR1::TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrate the oncogenic transcriptional signature of ASPSCR1::TFE3, by facilitating assembly of higher-order chromatin conformation structures demonstrated by HiChIP. Finally, ASPSCR1::TFE3 and VCP demonstrate co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP’s potential as a novel therapeutic target. VCP/p97 is identified as a co-factor to the fusion oncoprotein ASPSCR1::TFE3. They co-localize on chromatin, co-dependent for enhancer looping and transcriptional regulation in alveolar soft part sarcomas and Xp11- rearranged renal cell carcinomas.

Background: Triple-negative breast cancer (TNBC) is a subtype of aggressive breast cancer that lacks estrogen, progesterone and HER2 receptors. Consequently, chemotherapy is one of the main treatment options of TNBC. Macroautophagy (hereafter autophagy) is a catabolic pathway where lysosomal degradation of organelles and proteins provides nutrients that support cellular functions. Previous work demonstrated pro-survival roles of autophagy in TNBC and precipitated investigations to evaluate whether concurrent lysosomal inhibition improves chemotherapy efficacy. As lysosome and autophagy machinery interact extensively with the endocytic pathway that gives rise to exosomes, a type of extracellular vesicle of relevance in cancer, we investigated the effect of lysosomal inhibition on the content and function of TNBC-derived exosomes. Methods: TNBC cell line conditioned media was pre-cleared using differential centrifugation and concentrated using centrifugal filtration. The ExoQuick reagent was used to precipitate exosomes. western blotting, NanoSight, and transmission electron microscopy were used to characterize the exosomes isolated. Mass spectrometry was used to identify exosomal proteins. Proliferation and tube-formation of endothelial (HMEC-1) cells treated with TNBC-derived exosomes were measured as surrogate markers of angiogenesis. Results: Treatment of TNBC cell lines with lysosomal inhibitor chloroquine (CQ) blocked autophagy turnover and altered exosome biogenesis. CQ-treated TNBC produced fewer but more protein-rich exosomes compared to the controls. CQ treatment also altered the level of exosomal autophagy-related proteins. Exosomes derived from control and CQ-treated TNBC cells had different effects on HMEC-1 growth and tube formation. Summary/Conclusion: Perturbation of lysosomal physiology can impact both macroautophagy and exosomal cargo and function. Presence of autophagy-related proteins suggests the potential involvement of autophagy machinery in exosome biogenesis.

Background: Tumour cells have unique survival capacities in the GILA (growth in low attachment) condition, a stressful 3D culture condition in which non-tumour cells undergo apoptosis. However, the underlying molecular mechanism remains elusive. Since acute changes in mRNA translation represent a major component of stress adaptation, we hypothesized that tumour cells adapt to the GILA adversity via altering the translatome or the acute synthesis of specific proteins. Methods: Using Click technology coupled with pulsed-SILAC and mass spectrometry (Click-pSILAC), the global nascent protein translation profiles were compared between non-transformed and oncogene-transformed cells in the GILA condition. Moreover, the impact of acute translatome rewiring on the global proteome was also evaluated by Tandem Mass Tag labelling. The impact of Myo1b depletion on the global secretome of tumour cells was determined using the ClickpSILAC approach. Ultracentrifugation was used for exosome isolation, and NanoSight tracking analysis and electron microscopy were performed to quantify and visualize exosomes. Results: These systematic analyses uncovered that numerous key components of the membrane trafficking and exosome biogenesis pathway are upregulated in oncogene-transformed cells, such as Rabgef1, Sec23b, Myo1b and multiple Rab family proteins. In support of the role of this pathway in exosome biogenesis, transformed cells acquired increased exosome formation capacity compared with non-transformed cells. Importantly, exosomes derived from transformed cells could confer non-transformed cells with acquired adaptability in the GILA condition. Moreover, blocking Rab27b, one of the key regulators of exosome formation, diminished the stress adaptability and tumourigenicity of transformed cells. Furthermore, global secretome analyses uncovered that Myo1b depletion in tumour cells markedly decreased the abundance of exosome markers in the secretome, which has enabled us to identify Myo1b as a novel regulator of exosome biogenesis. Summary/Conclusion: Collectively, our findings suggest that oncogenes systematically reprogramme the membrane trafficking and exosome biogenesis pathway to promote stress adaptation and drive tumourigenicity in tumour cells.

The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.The t(X,17) chromosomal translocation, generating the ASPSCR1-TFE3 fusion oncoprotein, is the singular genetic driver of alveolar soft part sarcoma (ASPS) and some Xp11-rearranged renal cell carcinomas (RCC), frustrating efforts to identify therapeutic targets for these rare cancers. Proteomic analysis showed that VCP/p97, an AAA+ ATPase with known segregase function, was strongly enriched in co-immunoprecipitated nuclear complexes with ASPSCR1-TFE3. We demonstrate that VCP is a likely obligate co-factor of ASPSCR1-TFE3, one of the only such fusion oncoprotein co-factors identified in cancer biology. Specifically, VCP co-distributed with ASPSCR1-TFE3 across chromatin in association with enhancers genome-wide. VCP presence, its hexameric assembly, and its enzymatic function orchestrated the oncogenic transcriptional signature of ASPSCR1-TFE3, by facilitating assembly of higher-order chromatin conformation structures as demonstrated by HiChIP. Finally, ASPSCR1-TFE3 and VCP demonstrated co-dependence for cancer cell proliferation and tumorigenesis in vitro and in ASPS and RCC mouse models, underscoring VCP's potential as a novel therapeutic target.

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequence data from 1,727 unique individual AML patients, which revealed mutations in ubiquitin ligase family genes in 11.2% of adult AML samples with mutual exclusivity. The Skp1/Cul1/Fbox (SCF) E3 ubiquitin ligase complex gene FBXO11 was the most significantly downregulated gene of the SCF complex in AML. FBXO11 catalyzes K63-linked ubiquitination of a novel target, LONP1, which promotes entry into mitochondria, thereby enhancing mitochondrial respiration. Reduced mitochondrial respiration secondary to FBXO11 depletion imparts myeloid-biased stem cell properties in primary CD34+ hematopoietic stem progenitor cells (HSPC). In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRASG12D to generate serially transplantable AML enriched for primitive cells. Our findings suggest that reduced FBXO11 primes HSPC for myeloid-biased self-renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. Analysis of sequencing data from 1,727 unique AML patients revealed frequent mutations in ubiquitin ligase family genes. Loss of function of the Skp1/Cul1/Fbox (SCF) E3 ubiquitin ligase complex genes are found in 8 - 9% of adult AML patients including recurrent mutations in FBXO11. FBXO11 is the most significantly downregulated gene of the SCF complex in AML samples. Depletion of Fbxo11 promotes myeloid-biased stem cell maintenance and cooperates with AML1-ETO and mutant KRAS to generate serially transplantable mouse and human AML in in vivo models. FBXO11 mediates K63-linked polyubiquitination of the LONP1 mitochondrial protease, and loss of FBXO11 impairs LONP1 activity thereby reducing mitochondrial membrane potential, imparting stem cell properties and driving leukemogenesis. Our findings suggest that loss of FBXO11 function primes HSPC for myeloid-biased self-renewal through attenuation of LONP1-mediated regulation of mitochondrial function. Competing Interest Statement The authors have declared no competing interest. Footnotes * Number of patients included in the analyses (Added 100 new patients to bring total to 1727). This led us to revise figures 1, supplemental figure 1, and supplemental figure 6 to reflect the new patients inclusion in our datasets.