Tradeoff between metabolic i-proteasome addiction and immune evasion in triple-negative breast cancer.

In vitro studies have suggested proteasome inhibitors could be effective in triple-negative breast cancer (TNBC). We found that bortezomib and carfilzomib induce proteotoxic stress and apoptosis via the unfolded protein response (UPR) in TNBC cell lines, with sensitivity correlated with expression of immuno-(PSMB8/9/10) but not constitutive-(PSMB5/6/7) proteasome subunits. Equally, the transcriptomes of i-proteasome-high human TNBCs are enriched with UPR gene sets, and the genomic copy number landscape reflects positive selection pressure favoring i-proteasome activity, but in the setting of adjuvant treatment, this is actually associated with favorable prognosis. Tumor expression of PSMB8 protein (ß5i) is associated with levels of MHC-I, interferon-γ-inducible proteasome activator PA28ß, and the densities of stromal antigen-presenting cells and lymphocytes (TILs). Crucially, TILs were protective among TNBCs that maintain high ß5i but did not stratify survival amongst ß5i-low TNBCs. Moreover, ß5i expression was lower in brain metastases than in patient-matched primary breast tumors (n = 34; P = 0.007), suggesting that suppression contributes to immune evasion and metastatic progression. Hence, inhibiting proteasome activity could be counterproductive in the adjuvant treatment setting because it potentiates anti-TNBC immunity.

Mammary-specific ablation of Cyp24a1 inhibits development, reduces proliferation and increases sensitivity to vitamin D.

Active vitamin D (1,25(OH)2D) has been shown to regulate numerous cell processes in mammary cells. Degradation of 1,25(OH)2D is initiated by the mitochondrial enzyme, 25-hydroxyvitamin D 24-hydroxylase (CYP24 A1), and provides local control of 1,25(OH)2D bioactivity. Several reports of the association between elevated CYP24 A1 activity and breast cancer incidence, suggest that CYP24 A1 may be a target for therapeutic intervention. Whether CYP24 A1 activity within the mammary epithelium regulates 1,25(OH)2D levels and mammary gland development is yet to shown. We have used a conditional knockout of the Cyp24a1 gene specifically in the mammary epithelium to demonstrate reduced terminal end bud number, ductal outgrowth and branching during puberty and alveologenesis at early pregnancy, by inhibiting proliferation but not apoptosis in both basal and luminal MECs. In vitro study showed increased sensitivity of luminal MECs to lower levels of 1,25(OH)2D with the ablation of Cyp24a1 activity. In summary, Cyp24a1 within MECs plays an important role in modulating postnatal and pregnancy-associated mammary gland development which provides support for inhibiting CYP24 A1 as a potential approach to activating the vitamin D pathway in breast cancer prevention and therapy.

Therapeutic Targeting of KDM1A/LSD1 in Ewing Sarcoma with SP-2509 Engages the Endoplasmic Reticulum Stress Response.

Multi-agent chemotherapeutic regimes remain the cornerstone treatment for Ewing sarcoma, the second most common bone malignancy diagnosed in pediatric and young adolescent populations. We have reached a therapeutic ceiling with conventional cytotoxic agents, highlighting the need to adopt novel approaches that specifically target the drivers of Ewing sarcoma oncogenesis. As KDM1A/lysine-specific demethylase 1 (LSD1) is highly expressed in Ewing sarcoma cell lines and tumors, with elevated expression levels associated with worse overall survival (P = 0.033), this study has examined biomarkers of sensitivity and mechanisms of cytotoxicity to targeted KDM1A inhibition using SP-2509 (reversible KDM1A inhibitor). We report, that innate resistance to SP-2509 was not observed in our Ewing sarcoma cell line cohort (n = 17; IC50 range, 81 -1,593 nmol/L), in contrast resistance to the next-generation KDM1A irreversible inhibitor GSK-LSD1 was observed across multiple cell lines (IC50 > 300 µmol/L). Although TP53/STAG2/CDKN2A status and basal KDM1A mRNA and protein levels did not correlate with SP-2509 response, induction of KDM1B following SP-2509 treatment was strongly associated with SP-2509 hypersensitivity. We show that the transcriptional profile driven by SP-2509 strongly mirrors KDM1A genetic depletion. Mechanistically, RNA-seq analysis revealed that SP-2509 imparts robust apoptosis through engagement of the endoplasmic reticulum stress pathway. In addition, ETS1/HIST1H2BM were specifically induced/repressed, respectively following SP-2509 treatment only in our hypersensitive cell lines. Together, our findings provide key insights into the mechanisms of SP-2509 cytotoxicity as well as biomarkers that can be used to predict KDM1A inhibitor sensitivity in Ewing sarcoma. Mol Cancer Ther; 17(9); 1902-16. ©2018 AACR.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

Vitamin D3 signaling and breast cancer: Insights from transgenic mouse models.

The biologically active form of vitamin D3 (1,25(OH)2D) regulates epithelial cell differentiation, proliferation, and apoptosis, lending weight to clinical evidence linking vitamin D3 insufficiency to breast cancer incidence and mortality. Local dysregulation of vitamin D3 metabolism has been identified in patients with breast cancer, implying that disruption of 1,25(OH)2D signaling may contribute to breast cancer development in an autocrine or paracrine manner. Mouse mammary glands express the critical enzymes responsible for 1,25(OH)2D synthesis (Cyp2r1 and Cyp27b1), degradation (Cyp24a1), as well as the vitamin D3 receptor (Vdr), and genetically modified mouse models have revealed a great deal about the role of vitamin D3 in cancer initiation and progression. Ablation of Vdr or Cyp27b1 in murine models of mammary cancer reduces the anti-tumor effects of vitamin D3, while elevation of Cyp24a1 levels increases degradation of 1,25(OH)2D, leading to diminished anti-tumor effects. This review discusses the recent transgenic mouse models of vitamin D3 metabolism and the Vdr signaling network, and how these contribute to mammary gland development, and cancer tumorigenesis and progression. Collectively, these mouse models have helped clarify mechanisms of action of vitamin D3 signaling and suggest that activation or restoration of the vitamin D3 regulated pathway is a potential approach for human breast cancer prevention.

Naturally existing isoforms of miR-222 have distinct functions.

Deep-sequencing reveals extensive variation in the sequence of endogenously expressed microRNAs (termed 'isomiRs') in human cell lines and tissues, especially in relation to the 3' end. From the immunoprecipitation of the microRNA-binding protein Argonaute and the sequencing of associated small RNAs, we observe extensive 3'-isomiR variation, including for miR-222 where the majority of endogenously expressed miR-222 is extended by 1-5 nt compared to the canonical sequence. We demonstrate this 3' heterogeneity has dramatic implications for the phenotype of miR-222 transfected cells, with longer isoforms promoting apoptosis in a size (but not 3' sequence)-dependent manner. The transfection of longer miR-222 isomiRs did not induce an interferon response, but did downregulate the expression of many components of the pro-survival PI3K-AKT pathway including PIK3R3, a regulatory subunit whose knockdown phenocopied the expression of longer 222 isoforms in terms of apoptosis and the inhibition of other PI3K-AKT genes. As this work demonstrates the capacity for 3' isomiRs to mediate differential functions, we contend more attention needs to be given to 3' variance given the prevalence of this class of isomiR.

Azobenzene-containing photoswitchable proteasome inhibitors with selective activity and cellular toxicity.

A series of azobenzene-containing peptidic boronate esters was prepared and the activity of the thermally adapted states (TAS), enriched in trans isomer, and the photostationary states (PSS), enriched in cis isomer, for each compound were evaluated against ß5 and ß1 proteasome subunits. Compounds with a sterically demanding phenyl-substituted azobenzene at P2 (4c), and a less sterically demanding unsubstituted azobenzene at the N-terminus (5a), showed the greatest difference in activity between the two states. In both cases, the more active trans-enriched TAS had activity comparable to bortezomib and delanzomib. Furthermore, cis-enriched 4c inhibited tumor growth in both breast and colorectal carcinoma cell lines. Significantly, the initial trans-enriched TAS of 4c was not cytotoxic against the non-malignant MCF-10A cells.

MiR-766 induces p53 accumulation and G2/M arrest by directly targeting MDM4.

p53, a transcription factor that participates in multiple cellular functions, is considered the most important tumor suppressor. Previous evidence suggests that post-transcriptional deregulation of p53 by microRNAs contributes to tumorigenesis, tumor progression and therapeutic resistance. In the present study, we found that the microRNA miR-766 was aberrantly expressed in breast cancer, and that over-expression of miR-766 caused accumulation of wild-type p53 protein in multiple cancer cell lines. Supporting its role in the p53 signalling pathway, miR-766 decreased cell proliferation and colony formation in several cancer cell lines, and cell cycle analyses revealed that miR-766 causes G2 arrest. At a mechanistic level, we demonstrate that miR-766 enhances p53 signalling by directly targeting MDM4, an oncogene and negative regulator of p53. Analysis of clinical genomic data from multiple cancer types supports the relevance of miR-766 in p53 signalling. Collectively, our study demonstrates that miR-766 can function as a novel tumor suppressor by enhancing p53 signalling.

New Peptidomimetic Boronates for Selective Inhibition of the Chymotrypsin-like Activity of the 26S Proteasome.

Proteasome is a large proteinase complex that degrades proteins via its three catalytic activities. Among these activities, the "chymotrypsin-like" activity has emerged as the focus of drug discovery in cancer therapy. Here, we report new peptidomimetic boronates that are highly specific for the chymotrypsin-like catalytic activity of the proteasome. These new specific proteasome inhibitors were demonstrated to have higher in vitro potency and selective cytotoxicity for cancer cells compared to benchmark proteasome inhibitors: bortezomib and carfilzomib. In breast cancer cell lines, treatment with 1a or 2a induced accumulation of the high molecular weight polyubiqutinated proteins at similar levels observed for bortezomib and carfilzomib, indicating that cancer cell death caused by 1a/2a is chiefly due to proteasome inhibition.

Cancer Detection in Human Tissue Samples Using a Fiber-Tip pH Probe.

Intraoperative detection of tumorous tissue is an important unresolved issue for cancer surgery. Difficulty in differentiating between tissue types commonly results in the requirement for additional surgeries to excise unremoved cancer tissue or alternatively in the removal of excess amounts of healthy tissue. Although pathologic methods exist to determine tissue type during surgery, these methods can compromise postoperative pathology, have a lag of minutes to hours before the surgeon receives the results of the tissue analysis, and are restricted to excised tissue. In this work, we report the development of an optical fiber probe that could potentially find use as an aid for margin detection during surgery. A fluorophore-doped polymer coating is deposited on the tip of an optical fiber, which can then be used to record the pH by monitoring the emission spectra from this dye. By measuring the tissue pH and comparing with the values from regular tissue, the tissue type can be determined quickly and accurately. The use of a novel lift-and-measure technique allows for these measurements to be performed without influence from the inherent autofluorescence that commonly affects fluorescence-based measurements on biological samples. The probe developed here shows strong potential for use during surgery, as the probe design can be readily adapted to a low-cost portable configuration, which could find use in the operating theater. Use of this probe in surgery either on excised or in vivo tissue has the potential to improve success rates for complete removal of cancers. Cancer Res; 76(23); 6795-801. ©2016 AACR.

Identification of vitamin D3 target genes in human breast cancer tissue.

Multiple epidemiological studies have shown that high vitamin D3 status is strongly associated with improved breast cancer survival. To determine the molecular pathways influenced by 1 alpha, 25-dihydroxyvitamin D3 (1,25D) in breast epithelial cells we isolated RNA from normal human breast and cancer tissues treated with 1,25D in an ex vivo explant system. RNA-Seq revealed 523 genes that were differentially expressed in breast cancer tissues in response to 1,25D treatment, and 127 genes with altered expression in normal breast tissues. GoSeq KEGG pathway analysis revealed 1,25D down-regulated cellular metabolic pathways and enriched pathways involved with intercellular adhesion. The highly 1,25D up-regulated target genes CLMN, SERPINB1, EFTUD1, and KLK6were selected for further analysis and up-regulation by 1,25D was confirmed by qRT-PCR analysis in breast cancer cell lines and in a subset of human clinical samples from normal and cancer breast tissues. Ketoconazole potentiated 1,25D-mediated induction of CLMN, SERPINB1, and KLK6 mRNA through inhibition of 24-hydroxylase (CYP24A1) activity. Elevated expression levels of CLMN, SERPINB1, and KLK6 are associated with prolonged relapse-free survival for breast cancer patients. The major finding of the present study is that exposure of both normal and malignant breast tissue to 1,25D results in changes in cellular adhesion, metabolic pathways and tumor suppressor-like pathways, which support epidemiological data suggesting that adequate vitamin D3 levels may improve breast cancer outcome.

p53 Represses the Oncogenic Sno-MiR-28 Derived from a SnoRNA.

p53 is a master tumour repressor that participates in vast regulatory networks, including feedback loops involving microRNAs (miRNAs) that regulate p53 and that themselves are direct p53 transcriptional targets. We show here that a group of polycistronic miRNA-like non-coding RNAs derived from small nucleolar RNAs (sno-miRNAs) are transcriptionally repressed by p53 through their host gene, SNHG1. The most abundant of these, sno-miR-28, directly targets the p53-stabilizing gene, TAF9B. Collectively, p53, SNHG1, sno-miR-28 and TAF9B form a regulatory loop which affects p53 stability and downstream p53-regulated pathways. In addition, SNHG1, SNORD28 and sno-miR-28 are all significantly upregulated in breast tumours and the overexpression of sno-miR-28 promotes breast epithelial cell proliferation. This research has broadened our knowledge of the crosstalk between small non-coding RNA pathways and roles of sno-miRNAs in p53 regulation.

XI-006 induces potent p53-independent apoptosis in Ewing sarcoma.

There is an imperious need for the development of novel therapeutics for the treatment of Ewing sarcoma, the second most prevalent solid bone tumour observed in children and young adolescents. Recently, a 4-nitrobenzofuroxan derivative, XI-006 (NSC207895) was shown to diminish MDM4 promoter activity in breast cancer cell lines. As amplification of MDM4 is frequently observed in sarcomas, this study examined the therapeutic potential of XI-006 for the treatment of Ewing and osteosarcoma. XI-006 treatment of Ewing and osteosarcoma cell lines (n = 11) resulted in rapid and potent apoptosis at low micro-molar concentrations specifically in Ewing sarcoma cell lines (48 hr IC50 0.099-1.61 µM). Unexpectedly, apoptotic response was not dependent on MDM4 mRNA/protein levels or TP53 status. Alkaline/neutral comet and γH2AX immunofluorescence assays revealed that the cytotoxic effects of XI-006 could not be attributed to the induction of DNA damage. RNA expression analysis revealed that the mechanism of action of XI-006 could be accredited to the inhibition of cell division and cycle regulators such as KIF20A and GPSM2. Finally, potent synergy between XI-006 and olaparib (PARP inhibitor) were observed due to the down-regulation of Mre11. Our findings suggest that XI-006 represents a novel therapeutic intervention for the treatment of Ewing sarcoma.

Ankrd11 is a chromatin regulator involved in autism that is essential for neural development.

Ankrd11 is a potential chromatin regulator implicated in neural development and autism spectrum disorder (ASD) with no known function in the brain. Here, we show that knockdown of Ankrd11 in developing murine or human cortical neural precursors caused decreased proliferation, reduced neurogenesis, and aberrant neuronal positioning. Similar cellular phenotypes and aberrant ASD-like behaviors were observed in Yoda mice carrying a point mutation in the Ankrd11 HDAC-binding domain. Consistent with a role for Ankrd11 in histone acetylation, Ankrd11 was associated with chromatin and colocalized with HDAC3, and expression and histone acetylation of Ankrd11 target genes were altered in Yoda neural precursors. Moreover, the Ankrd11 knockdown-mediated decrease in precursor proliferation was rescued by inhibiting histone acetyltransferase activity or expressing HDAC3. Thus, Ankrd11 is a crucial chromatin regulator that controls histone acetylation and gene expression during neural development, thereby providing a likely explanation for its association with cognitive dysfunction and ASD.

Characterization of ANKRD11 mutations in humans and mice related to KBG syndrome.

Mutations in ANKRD11 have recently been reported to cause KBG syndrome, an autosomal dominant condition characterized by intellectual disability (ID), behavioral problems, and macrodontia. To understand the pathogenic mechanism that relates ANKRD11 mutations with the phenotype of KBG syndrome, we studied the cellular characteristics of wild-type ANKRD11 and the effects of mutations in humans and mice. We show that the abundance of wild-type ANKRD11 is tightly regulated during the cell cycle, and that the ANKRD11 C-terminus is required for the degradation of the protein. Analysis of 11 pathogenic ANKRD11 variants in humans, including six reported in this study, and one reported in the Ankrd11 (Yod/+) mouse, shows that all mutations affect the C-terminal regions and that the mutant proteins accumulate aberrantly. In silico analysis shows the presence of D-box sequences that are signals for proteasome degradation. We suggest that ANKRD11 C-terminus plays an important role in regulating the abundance of the protein, and a disturbance of the protein abundance due to the mutations leads to KBG syndrome.

The NF1 gene revisited - from bench to bedside.

Neurofibromatosis type 1 (NF1) is a relatively common tumour predisposition syndrome related to germline aberrations of NF1, a tumour suppressor gene. The gene product neurofibromin is a negative regulator of the Ras cellular proliferation pathway, and also exerts tumour suppression via other mechanisms. Recent next-generation sequencing projects have revealed somatic NF1 aberrations in various sporadic tumours. NF1 plays a critical role in a wide range of tumours. NF1 alterations appear to be associated with resistance to therapy and adverse outcomes in several tumour types. Identification of a patient's germline or somatic NF1 aberrations can be challenging, as NF1 is one of the largest human genes, with a myriad of possible mutations. Epigenetic factors may also also contribute to inadequate levels of neurofibromin in cancer cells. Clinical trials of NF1-based therapeutic approaches are currently limited. Preclinical studies on neurofibromin-deficient malignancies have mainly been on malignant peripheral nerve sheath tumour cell lines or xenografts derived from NF1 patients. However, the emerging recognition of the role of NF1 in sporadic cancers may lead to the development of NF1-based treatments for other tumour types. Improved understanding of the implications of NF1 aberrations is critical for the development of novel therapeutic strategies.

SCF-FBXO31 E3 ligase targets DNA replication factor Cdt1 for proteolysis in the G2 phase of cell cycle to prevent re-replication.

FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.

Nutlin-3a efficacy in sarcoma predicted by transcriptomic and epigenetic profiling.

Nutlin-3a is a small-molecule antagonist of p53/MDM2 that is being explored as a treatment for sarcoma. In this study, we examined the molecular mechanisms underlying the sensitivity of sarcomas to Nutlin-3a. In an ex vivo tissue explant system, we found that TP53 pathway alterations (TP53 status, MDM2/MDM4 genomic amplification/mRNA overexpression, MDM2 SNP309, and TP53 SNP72) did not confer apoptotic or cytostatic responses in sarcoma tissue biopsies (n = 24). Unexpectedly, MDM2 status did not predict Nutlin-3a sensitivity. RNA sequencing revealed that the global transcriptomic profiles of these sarcomas provided a more robust prediction of apoptotic responses to Nutlin-3a. Expression profiling revealed a subset of TP53 target genes that were transactivated specifically in sarcomas that were highly sensitive to Nutlin-3a. Of these target genes, the GADD45A promoter region was shown to be hypermethylated in 82% of wild-type TP53 sarcomas that did not respond to Nutlin-3a, thereby providing mechanistic insight into the innate ability of sarcomas to resist apoptotic death following Nutlin-3a treatment. Collectively, our findings argue that the existing benchmark biomarker for MDM2 antagonist efficacy (MDM2 amplification) should not be used to predict outcome but rather global gene expression profiles and epigenetic status of sarcomas dictate their sensitivity to p53/MDM2 antagonists.

TAp63 regulates oncogenic miR-155 to mediate migration and tumour growth.

miR-155 is an oncogenic microRNA which is upregulated in many solid cancers. The targets of miR-155 are well established , with over 100 confirmed mRNA targets. However, the regulation of miR-155 and the basis of its upregulation in cancer is not well understood. We have previously shown that miR-155 is regulated by p63, and here we investigate the role of the major p63 isoforms TAp63 and ΔNp63 in this regulation. When the TAp63 isoform was knocked down, or exogenously overexpressed, miR-155 levels were elevated in response to TAp63 knockdown or reduced in response to TAp63 overexpression. The ΔNp63 isoform is shown to directly bind to the p63 response element on the miR-155 host gene, and this binding is enriched when TAp63 is knocked down. This could indicate that TAp63 prevents ΔNp63 from binding to the miR-155 host gene. The knockdown of TAp63, and the subsequent elevation of miR-155, enhances migration and tumour growth similar to that seen when directly overexpressing miR-155. The migratory phenotype is abrogated when miR-155 is inhibited, indicating that miR-155 is responsible for the phenotypic effect of TAp63 knockdown.