BRCA1 basaalisen rintasyövän synnyssä ja hoidossa

Women with germline mutations of BRCA1 gene are predisposed to the development of basal-like breast cancer, which is characterized by the absence of the hormonal and growth factor receptors, ER, PR and HER2. Mutation of BRCA1 predicts increased sensitivity to certain DNA-damaging agents, e.g. PARP inhibitors and cisplatin. Yet in clinical trials, some BRCA1-mutant breast tumors show resistance to these drugs. Therefore, it is necessary to understand the molecular mechanisms of the acquired resistance and identify novel therapeutic targets for the treatment of those tumors. In order to better understand the biology of breast cancers caused by BRCA1 mutations, we collected and characterized four BRCA1-mutant breast cancer cell lines as surrogates of BRCA1-mutant tumors. Together with several cell lines expressing wildtype BRCA1, we tested their sensitivities to a panel of DNA-damaging agents. We also carried out a high-throughput chemical compound screen on the BRCA1-mutant and BRCA1-wildtype cell lines, and performed a proteome profiling assay to test the kinase activities of those cell lines. Our results reveal extensive heterogeneity among the BRCA1-mutant breast cancer cell lines, which showed resistance to DNA-damaging agents. Then we created isogenic MDA-MB-231 cells with or without BRCA1 depletion by siRNA transfection, and carried out a high-throughput chemical compound screen on the pair of cells in order to identify potential targets that are synthetically lethal with BRCA1 deficiency. Two proteasome inhibitors, bortezomib and carfilzomib, were found to be able to selectively kill BRCA1-depleted cells. Further studies on mechanisms demonstrate that the proteasome inhibitor bortezomib does not induce DNA damage; rather it inactivates G1 cell cycle checkpoint in BRCA1-deficient cells and leads to the accumulation of these cells at G2/M phase. This is caused by inactivation of the retinoblastoma protein (Rb) through its hyperphosphorylation, which in turn activates its downstream transcription factor E2F1. In addition, bortezomib causes compromised G2/M cell cycle checkpoint in BRCA1-deficient cells, which drives cells to enter mitosis and leads to apoptosis due to uncontrolled cell division. In order to investigate the mechanisms underlying the consistent basal-like phenotype of breast tumors associated with BRCA1 mutations, we established an in vitro assay to study the transition of mouse mammary epithelial cells from luminal to basal lineages. Our results showed that loss of BRCA1 promotes basal-like differentiation by sustaining ΔNp63 activity. In luminal cells, ΔNp63 is not expressed or remains inactive through localization in nucleoli. Depletion of BRCA1 leads to translocation of ΔNp63 into nucleoplasm and promotes transition of luminal cells into a basal state. This study provides a potential link between BRCA1 loss and the basal-like differentiation, which may help to explain why BRCA1-mutant breast cancer tends to bear a basal-like phenotype.Naiset, jotka kantavat somaattista BRCA1 mutaatiota, ovat alttiita basaalisen rintasyövän synnylle. Tälle syövälle on tyypillistä hormooni-ja kasvutekijäreseptoreiden (ER, PR, Her2) puuttuminen. BRCA1 geenimutaatioiden on havaittu herkistävän syöpäsoluja tietyille DNA vaurioita-aiheuttaville yhdisteille kuten PARP estäjäille ja cisplatiinille. Kliiniset lääkekokeilut ovat kuitenkin paljastaneet osan BRCA1 mutatoituneista kasvaimista vastustuskykyisiksi näille lääkeaineille. Vastustuskyvyn taustalla olevien molekyylitason mekanismien ymmärtäminen on siten ensisijaisen tärkeää uusien lääkekohteiden löytämiselle. Jotta pystyttäisiin paremmin ymmärtämään BRCA1 mutatoituneen rintasyövän biologiaa, tässä väitöskirjatyössä karakterisoitiin neljä BRCA1 mutatoitunutta rintasyöpäsolulinjaa yhdessä usean normaalia BRCA1 geeniä ilmentävien solulinjan kanssa ja seurattiin niiden vastetta DNA-vaurioita aiheuttaville yhdisteille. Lisäksi työssä seulottiin kemiallisilla yhdisteillä ne solulinjat, joille saatiin vaste. Seulottujen solulinjojen vastetta ja kinaasiaktiivisuutta mitattiin proteiinien profilointimenetelmällä. Tuloksemme osoittivat laajamittaista heterogeenisyyttä BRCA1 mutatoituneiden solulinjojen herkkyydessä DNA- vaurioita aiheuttaville yhdisteille. Seuraavaksi hiljensimme MDA-MB-231 solulinjasta BRCA1 geenin siRNA transfektiolla ja solulinjan muuttunutta herkkyyttä eri yhdisteille testattiin kemikaaliseulonnalla. Erot saatiin vertaamalla BRCA1 hiljennettyä linjaa alkuperäiseen BRCA1 ilmentävään MDA-MB-231 kantaan. Tarkoituksena oli löytää mahdollisia uusia lääkekohteita, jotka yhdessä BRCA1 mutaation kanssa johtaisivat solukuolemaan. Seulonnassa löydettiin kaksi proteasomiestäjää bortezomib ja carfilzomib, jotka pystyivät valikoivasti tappamaan BRCA1 mutatoidut solut. Mekanistiset jatkotutkimukset paljastivat, ettei bortezomib aiheuttanut DNA vaurioita vaan esti G1 solusylin pysäytysvaiheen BRCA1-puutteisissa soluissa ja johtaa näiden solujen kerääntymisen G2/M vaiheeseen. Tämä oli seurausta retinoblastoma proteiinin (Rb) inaktivaatiosta hyperfosforylaation kautta, joka vuorostaan aktivoi transkiptiotekijä E2F1n. Lisäksi bortezomib aiheutti osittaisen G2/M solusyklin pysäytyksen BRCA1 puutteisissa soluissa, joka ajoi solut mitoosiin ja edelleen ohjelmoituun solukuolemaan halitsemattoman solunjakautumisen seurauksena. Tutkiaksemme mekanismeja, jotka johtavat basaalisen rintasyövän syntyyn, pystytimme in vitro-menetelmän, jonka avulla seurasimme hiiren maitorauhasen primaarisolujen erilaistumista luminaalisesta basaaliseen ilmiasuun. Tuloksemme osoittavat BRCA1 puutoksen edistävän Np63 aktiivisuutta, joka on keskeinen tekijä solujen erilaistumisessa basaalisoluiksi. ΔNp63 ei ilmenny luminaalisoluissa tai proteiinia hajoitetaan jatkuvasti, minkä vuoksi sen nähdään lokalisoituvan tumajyväsiin. BRCA1 geenin poisto näytti johtavan ΔNp63 aktivoitumiseen ja lokalisoitumiseen tumaan, minkä seurauksena soluidentiteetti muuttui kohti basaalista. Tulokset selittänevät osaltaan mahdollisesti miksi BRCA1 mutatoituneilla rintasyövillä on usein basaalinen ilmiasu

AMPK, a Regulator of Metabolism and Autophagy, Is Activated by Lysosomal Damage via a Novel Galectin-Directed Ubiquitin Signal Transduction System

AMPK is a central regulator of metabolism and autophagy. Here we show how lysosomal damage activates AMPK. This occurs via a hitherto unrecognized signal transduction system whereby cytoplasmic sentinel lectins detect membrane damage leading to ubiquitination responses. Absence of Galectin 9 (Gal9) or loss of its capacity to recognize lumenal glycans exposed during lysosomal membrane damage abrogate such ubiquitination responses. Proteomic analyses with APEX2-Gal9 have revealed global changes within the Gal9 interactome during lysosomal damage. Gal9 association with lysosomal glycoproteins increases whereas interactions with a newly identified Gal9 partner, deubiquitinase USP9X, diminishes upon lysosomal injury. In response to damage, Gal9 displaces USP9X from complexes with TAK1 and promotes K63 ubiquitination of TAK1 thus activating AMPK on damaged lysosomes. This triggers autophagy and contributes to autophagic control of membrane-damaging microbe Mycobacterium tuberculosis. Thus, galectin and ubiquitin systems converge to activate AMPK and autophagy during endomembrane homeostasis

BRCA1-deficient breast cancer cell lines are resistant to MEK inhibitors and show distinct sensitivities to 6-thioguanine

Germ-line or somatic inactivation of BRCA1 is a defining feature for a portion of human breast cancers. Here we evaluated the anti-proliferative activity of 198 FDA-approved and experimental drugs against four BRCA1-mutant (HCC1937, MDA-MB-436, SUM1315MO2, and SUM149PT) and four BRCA1-wild-type (MDA-MB-231, SUM229PE, MCF10A, and MCF7) breast cancer cell lines. We found that all BRCA1-mutant cell lines were insensitive to inhibitors of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) Selumetinib and Pimasertib in contrast to BRCA1-wildtype control cell lines. However, unexpectedly, only two BRCA1-mutant cell lines, HCC1937 and MDA-MB-436, were hypersensitive to a nucleotide analogue 6-thioguanine (6-TG). SUM149PT cells readily formed radiation-induced RAD51-positive nuclear foci indicating a functional homologous recombination, which may explain their resistance to 6-TG. However, the reason underlying 6-TG resistance of SUM1315MO2 cells remains unclear. Our data reveal a remarkable heterogeneity among BRCA1-mutant cell lines and provide a reference for future studies.Peer reviewe

MERIT, a cellular system coordinating lysosomal repair, removal and replacement

Membrane integrity is essential for cellular survival and function. The spectrum of mechanisms protecting cellular and intracellular membranes is not fully known. Our recent work has uncovered a cellular system termed MERIT for lysosomal membrane repair, removal and replacement. Specifically, lysosomal membrane damage induces, in succession, ESCRT-dependent membrane repair, macroautophagy/autophagy-dominant removal of damaged lysosomes, and initiation of lysosomal biogenesis via transcriptional programs. The MERIT system is governed by galectins, a family of cytosolically synthesized lectins recognizing β-galactoside glycans. We found in this study that LGALS3 (galectin 3) detects membrane damage by detecting exposed lumenal glycosyl groups, recruits and organizes ESCRT components PDCD6IP/ALIX, CHMP4A, and CHMPB at damaged sites on the lysosomes, and facilitates ESCRT-driven repair of lysosomal membrane. At later stages, LGALS3 cooperates with TRIM16, an autophagy receptor-regulator, to engage autophagy machinery in removal of excessively damaged lysosomes. In the absence of LGALS3, repair and autophagy are less efficient, whereas TFEB nuclear translocation increases to compensate lysosomal deficiency via de novo lysosomal biogenesis. The MERIT system protects endomembrane integrity against a broad spectrum of agents damaging the endolysosomal network including lysosomotropic drugs, Mycobacterium tuberculosis, or neurotoxic MAPT/tau. Abbreviations: AMPK: AMP-activated protein kinase; APEX2: engineered ascorbate peroxidase 2; ATG13: autophagy related 13; ATG16L1: autophagy related 16 like 1; BMMs: bone marrow-derived macrophages; ESCRT: endosomal sorting complexes required for transport; GPN: glycyl-L-phenylalanine 2-naphthylamide; LLOMe: L-leucyl-L-leucine methyl ester; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERIT: membrane repair, removal and replacement; MTOR: mechanistic target of rapamycin kinase; TFEB: transcription factor EB; TFRC: transferrin receptor; TRIM16: tripartite motif-containing 16

Galectin-3 Coordinates a Cellular System for Lysosomal Repair and Removal

Jia et al. show that Galectin-3 recruits ESCRT components to damaged lysosomes for repair and restoration of their function. During sustained lysosomal injury, galectins induce autophagy and lysosomal biogenesis for a staged repair, removal, and replacement program. This response is deployed during damage with neurotoxic tau or Mycobacterium tuberculosis infection

TP53 supports basal-like differentiation of mammary epithelial cells by preventing translocation of deltaNp63 into nucleoli

Peer reviewe

The relationship between the chemical composition and the quality of the soybean film during peeling process

Soybean film is a traditional nonfermented soy product in China. It is a film formed on the surface of the soymilk during heating process. The nutrient components of soymilk will affect the quality of the soybean film. The results of this study showed that during the peeling process, the proportion of protein and carbohydrate in soymilk decreased, and the proportion of lipid increased. The mechanical properties (fracture extension and tensile strength) of the soybean film decreased during the peeling process. During the heating treatment, the Maillard reaction occurred and its intermediate products accumulated, which caused the change in soybean film color. White globular protein granules (<100 nm) existed on the surface of the soybean film. The lipid that was not wrapped by the protein network structure was exposed, and the evaporation of water led to the formation of black and gray holes on the skin (<500 nm). In addition, the results of correlation analysis showed that the changes in color, taste, and odor, as well as the mechanical properties of the skin, were all related to the changes in nutrients in the soybean film during peeling. This research provided a deeper understanding of the quality change in the soybean milk and the soybean film during the heating process

Characterization and phylogenetic analysis of the mitochondrial genome of Sarcocheilichthys sinensis (Bleeker) from Baima Hu Lake

Sarcocheilichthys sinensis (Bleeker), is a small-sized benthopelagic fish with ornamental value. In the present study, the complete mitochondrial genome of S. sinensis was sequenced and determined. The complete mitogenome of S. sinensis was 16,683 bp in length, consisting of 22 tRNA genes, 13 protein-coding genes, 2 rRNA genes, and 2 non-coding regions. The overall base composition of the S. sinensis mitogenome is 30.50% A, 26.28% T, 26.60% C, and 16.61% G, exhibiting obvious AT bias (56.79%). The phylogenetic analysis showed that S. sinensis clustered in genus Sarcocheilichthys. Present study provides useful data to population genetics and conservation biology of Sarcocheilichthys fishes

Lymphadenectomy and optimal excise lymph nodes count for early-stage primary fallopian tube cancer: a SEER-based study

Abstract Backgrounds There is still no consensus on the significance of Lymphadenectomy (LD) and the number of lymph nodes that need to be excised (ELNs) for adequate LD in patients with early-stage primary fallopian tube cancer (PFTC). Our endeavor is geared towards deepening comprehension of LD in early-stage PFTC and identify the optimal cut-off of ELNs. Methods This SEER-based study analyzed the clinical data of patients with early-stage PFTC between 2000 and 2018. X-tile was employed to confirm the optimal cut-off of ELNs. The survival data between groups were analyzed by the Kaplan-Meier estimates, Log-rank test and Cox proportional hazards model. Results There was significant improvement in both mean cancer-specific survival (CSS, p  11, n = 574) were significantly longer than these in inadequate LD group (ELNs ≤ 11, n = 738). Adequate LD, FIGO stage, tumor grade and histology were significant prognostic factors for CSS and OS. Conclusion LD is an independent protective prognostic factor of patients with early-stage PFTC. The association between ELNs > 11 and an improved prognosis is evident. Future studies are needed to further clarify the results above