Biology subjects

Ding, K.

Publications and source records attributed to Ding, K..

At least 55 records · Page 3Linked to original sources

bioRxiv2022

Sniff Olfactometer (SO) Protocols

Most olfactometers used to study human olfaction have stimulus durations of more than 1 second and often lasting minutes(Dravnieks 1975; Leland et al. 2001; Schmidt and Cain 2010). During long stimulations, olfactory receptor responses and their resulting behaviors are modulated by adaptation and habituation to the stimulus(Pellegrino et al. 2017; Rankin 2009; Wilson and Linster 2008). For example, EOG results from the first deorphanized olfactory receptor tissue reached a maximum in [~]1 s, dropping to 1/2 maximum in the next second, and showing little signal reduction until the stimulation stopped after 6 seconds(Zhao et al. 1998). Longer stimulations can result in complete habituation; receptors still respond even though the behavior shows complete habituation (Barwich 2014). To minimize the effects of adaption and habituation on stimulus responses, the sniff olfactometer (SO) combined the precision of a blast olfactometer with the gentleness of a stream olfactometer by blasting a brief odorant puff (70ms duration) into a subjects self-imposed inhalation air stream(Rochelle 2017; Rochelle et al. 2017b; Wyckoff and Acree 2017). Here we describe SO protocols for threshold determinations of odorants in aqueous headspaces using odorant recognition probabilities associated with Log(odorant-concentrations(Rochelle et al. 2017a)). During a single trial a subject, preconditioned to associate a veridical name with a given odor (e.g., a pyrazine with "nuts" when the odor was detected and "not nuts" if it wasnt) was cued to "inhale" and 750ms later, a 15ml-70ms puff of odorant headspace was delivered into their inhalation airstream. A session consisted of 12 randomized double-blind trials of 3 different odorant concentrations. Additional sessions with different concentrations were conducted until the response probability to the samples ranged from below 0.2 to above 0.8. The robustness of the fitted function and the size of their confidence intervals depended on the difference between the concentrations of the odorants during a single session: small differences in sample concentration resulted in the data failing to fit a logistic function; larger concentration differences resulted in a better fit to the model. However, if one of the stimuli had no odorant at all i.e., a blank, the response to the blank was random.

animal behavior and cognition↗

bioRxiv2022

Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila

When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue- and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2000 genes and that approximately 40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase (Hipk) as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.

physiology↗

bioRxiv2022

Observing noncovalent interactions in experimental electron density for macromolecular systems: A novel perspective for protein-ligand interaction research

We report for the first time the use of experimental electron density (ED) in the Protein Data Bank for modeling non-covalent interactions (NCIs) for protein-ligand complexes. Our methodology is based on the reduced electron density gradient (RDG) theory describing intermolecular NCI by ED and its first derivative. We established a database called the Experimental NCI Database (ExptNCI; http://ncidatabase.stonewise.cn/#/nci) containing ED saddle points, indicating ~200,000 NCIs from over 12,000 protein-ligand complexes. We also demonstrated the use of the database for depicting amide-{pi} interactions in a protein-ligand binding system. In summary, the database provides details on experimentally observed NCIs for protein-ligand complexes and can support future studies, including studies on rarely documented NCIs and the development of artificial intelligent models for protein-ligand binding prediction.

biochemistry↗

bioRxiv2021

Coupling adipose tissue architecture and metabolism via cytoophidia

Tissue architecture determines its unique physiology and function. How these properties are intertwined has remained unclear. Here, we show that the metabolic enzyme CTP synthase (CTPS) form filamentous structures termed cytoophidia along the adipocyte cortex in Drosophila adipose tissue. Interestingly, loss of cytoophidia, whether due to reduced CTPS expression or a point mutation that specifically abrogates its polymerization ability, leads to downregulated Collagen-Integrin signaling, weakened adipocyte adhesion, and defective adipose architecture. Strikingly, CTPS specifically binds with Integrin subunit 2, which influences Integrin function and Collagen IV deposition. cytoophidia promote Collagen IV mRNA expression and thus its extracellular deposition to strengthen adipocyte adhesion. Remarkably, Collagen IV-Integrin signaling reciprocally regulates cytoophidium formation at a post-translational level. Together, we demonstrate that a positive feedback signaling loop containing both cytoophidia and Integrin adhesion complex couples tissue architecture and metabolism in the fly adipose.

developmental biology↗

bioRxiv2021

Novel pectin from crude polysaccharide of Syzygium aromaticum against SARS-CoV-2 activities by targeting 3CLpro

To date, COVID-19 is still a severe threat to public health, hence specific effective therapeutic drugs development against SARS-CoV-2 is urgent needed. 3CLpro and PLpro and RdRp are the enzymes required for the SARS-CoV-2 RNA synthesis. Therefore, binding to the enzyme may interfere the enzyme function. Before, we found that sulfated polysaccharide binding to 3CLpro might block the virus replication. Hence, we hypothesize that negative charged pectin glycan may also impede the virus replication. Here we show that 922 crude polysaccharide from Syzygium aromaticum may near completely block SARS-CoV-2 replication. The inhibition rate was 99.9% (EC50 : 0.90 M). Interestingly, 922 can associates with 3CLpro, PLpro and RdRp. We further show that the homogeneous glycan 922211 from 922 may specifically attenuate 3CL protease activity. The IC50s of 922 and 922211 against 3CLpro are 4.73 {+/-} 1.05 {micro}M and 0.18 {+/-} 0.01 {micro}M, respectively. Monosaccharide composition analysis reveals that 922211 with molecular weight of 78.7 kDa is composed of rhamnose, galacturonic acid, galactose and arabinose in the molar ratio of 8.21 : 37.81 : 3.58 : 4.49. The structure characterization demonstrated that 922211 is a homogalacturonan linked to RG-I pectin polysaccharide. The linear homogalacturonan part in the backbone may be partly methyl esterified while RG-I type part bearing 1, 4-linked -GalpA, 1, 4-linked -GalpAOMe and 1, 2, 4-linked -Rhap. There are four branches attached to C-1 or C4 position of Rhamnose glycosyl residues on the backbone. The branches are composed of 1, 3-linked {beta}-Galp, terminal (T)-linked {beta}-Galp, 1, 5-linked -Araf, T-linked -Araf, 4-linked -GalpA and/or 4-linked {beta}-GalpA. The above results suggest that 922 and 922211 might be a potential novel leading compound for anti-SARS-CoV-2 new drug development.

pharmacology and toxicology↗

bioRxiv2021

Structural characterization of cocktail-like targeting polysaccharides from Ecklonia kurome Okam and their anti-SARS-CoV-2 activities in vitro

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological agent responsible for the worldwide coronavirus disease 2019 (COVID-19) outbreak. Investigation has confirmed that polysaccharide heparan sulfate can bind to the spike protein and block SARS-CoV-2 infection. Theoretically, similar structure of nature polysaccharides may also have the impact on the virus. Indeed, some marine polysaccharide has been reported to inhibit SARS-Cov-2 infection in vitro, however the convinced targets and mechanism are still vague. By high throughput screening to target 3CLpro enzyme, a key enzyme that plays a pivotal role in the viral replication and transcription using nature polysaccharides library, we discover the mixture polysaccharide 375 from seaweed Ecklonia kurome Okam completely block 3Clpro enzymatic activity (IC50, 0.48 {micro}M). Further, the homogeneous polysaccharide 37502 from the 375 may bind to 3CLpro molecule well (kD value : 4.23 x 10-6). Very interestingly, 37502 also can potently disturb spike protein binding to ACE2 receptor (EC50, 2.01 {micro}M). Importantly, polysaccharide 375 shows good anti-SARS-CoV-2 infection activity in cell culture with EC50 values of 27 nM (99.9% inhibiting rate at the concentration of 20 {micro}g/mL), low toxicity (LD50: 136 mg/Kg on mice). By DEAE ion-exchange chromatography, 37501, 37502 and 37503 polysaccharides are purified from native 375. Bioactivity test show that 37501 and 37503 may impede SARS-Cov-2 infection and virus replication, however their individual impact on the virus is significantly less that of 375. Surprisingly, polysaccharide 37502 has no inhibition effect on SARS-Cov-2. The structure study based on monosaccharide composition, methylation, NMR spectrum analysis suggest that 375 contains guluronic acid, mannuronic acid, mannose, rhamnose, glucouronic acid, galacturonic acid, glucose, galactose, xylose and fucose with ratio of 1.86 : 9.56 : 6.81 : 1.69 : 1.00 : 1.75 : 1.19 : 11.06 : 4.31 : 23.06. However, polysaccharide 37502 is an aginate which composed of mannuronic acid (89.3 %) and guluronic acid (10.7 %), with the molecular weight (Mw) of 27.9 kDa. These results imply that mixture polysaccharides 375 works better than the individual polysaccharide on SARS-Cov-2 may be the cocktail-like polysaccharide synergistic function through targeting multiple key molecules implicated in the virus infection and replication. The results also suggest that 375 may be a potential drug candidate against SARS-CoV-2.

biochemistry↗

bioRxiv2020

ESR1 mutant breast cancers show elevated basal cytokeratins and immune activation

Estrogen receptor alpha (ER/ESR1) is mutated in 30-40% of endocrine resistant ER-positive (ER+) breast cancer. ESR1 mutations cause ligand-independent growth and increased metastasis in vivo and in vitro. Despite the distinct clinical features and changes in therapeutic response associated with ESR1 mutations, there are no data about their potential role in intrinsic subtype switching. Applying four luminal and basal gene set pairs, ESR1 mutant cell models and clinical samples showed a significant enrichment of basal subtype markers. Among them, the six basal cytokeratins (BCKs) were the most enriched genes. Induction of BCKs was independent of ER binding and instead associated with chromatin reprogramming centered around a progesterone receptor-orchestrated topological associated domain at the KRT14/16/17 genomic region. Unexpectedly, high BCK expression in ER+ primary breast cancer is associated with good prognosis, and these tumors show enriched activation of a number of immune pathways, a distinctive feature shared with ESR1 mutant tumors. S100A8 and S100A9 were among the most highly induced immune mediators shared between high-BCKs ER+ and ESR1 mutant tumors, and single-cell RNA-seq analysis inferred their involvement in paracrine crosstalk between epithelial and stromal cells. Collectively, these observations demonstrate that ESR1 mutant tumors gain basal features with induction of basal cytokeratins via epigenetic mechanisms in rare subpopulation of cells. This is associated with increased immune activation, encouraging additional studies of immune therapeutic vulnerabilities in ESR1 mutant tumors.

cancer biology↗

bioRxiv2020

Dispatched conformational dynamics couples transmembrane Na+ flux to release of lipid-modified Hedgehog signal

The DISP1 protein, related to the NPC1 and PTCH1 cholesterol transporters and to H+-driven transporters of the RND family, enables tissue patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly-localized sites of embryonic expression. A 2.5 [A] resolution cryo-EM structure of DISP1 revealed three Na+ ions coordinated within a channel that traverses its transmembrane domain. This channel and Na+-binding are disrupted in DISP1-NNN (3.4 [A] resolution), a variant with isosteric substitutions for three intramembrane aspartates that each coordinate and neutralize charge for one of the three Na+ ions. DISP1-NNN and other variants that singly disrupt the Na+ sites retain binding to the lipid-modified Hedgehog protein, but most fail to export Hedgehog. The Sonic hedgehog signal (ShhN) interacts with the DISP1 extracellular domains (2.7 [A] complex structure), including an unusual extended DISP1 arm, at a location well above the membrane. Variability analysis reveals a dynamic series of DISP1 conformations, only a restricted subset of which appear to bind ShhN. The bound and unbound DISP1 conformations display distinct Na+ site occupancies; these differences, in conjunction with the unusual locations of certain lipids bound to DISP1, suggest a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane, thus enabling its tissue patterning activity. The Na+-coordinating residues resolved in our DISP1 structures are wholly or partly conserved in some of the other metazoan RND family members, such as PTCH1 and NPC1, suggesting the utilization of Na+ flux to power their conformationally-driven activities.

biophysics↗

bioRxiv2020

Biomarkers of Memory Variability in Traumatic Brain Injury

Traumatic brain injury (TBI) is a leading cause of cognitive disability and is often associated with significant impairment in episodic memory. In TBI survivors, as in healthy controls, there is marked variability between individuals in memory ability. Using recordings from indwelling electrodes, we characterized and compared the oscillatory biomarkers of mnemonic variability in two cohorts of epilepsy patients: a group with a history of moderate-to-severe TBI (n = 37) and a group of non-TBI controls (n = 111) closely matched for demographics and electrode coverage. Analysis of these recordings demonstrated that increased high frequency power and decreased theta power across a broad set of brain regions mark periods of successful memory formation in both groups. As features in a logistic-regression classifier, spectral power biomarkers effectively predicted recall probability, with little difference between TBI and non-TBI controls. The two groups also displayed similar patterns of theta-frequency connectivity during successful encoding periods. These biomarkers of successful memory, highly conserved between TBI patients and controls, could serve as the basis for novel therapies that target disordered memory across diverse forms of neurological disease.

neuroscience↗

bioRxiv2020

Acquired mutations and transcriptional remodeling in long-term estrogen-deprived locoregional breast cancer recurrences

Endocrine therapy resistance is a hallmark of advanced estrogen receptor (ER) positive breast cancer. In this study, we performed DNA/RNA hybrid-capture sequencing on 12 locoregional recurrences after long-term estrogen-deprivation along with each tumors matched primary. Despite being up to 7 years removed from the primary lesion, most recurrences harbored similar intrinsic transcriptional and copy number profiles. Only two genes, AKAP9 and KMT2C, were found to have single nucleotide variant (SNV) enrichments in more than one recurrence. Enriched mutations in single cases included SNVs within transcriptional regulators such as ARID1A, TP53, FOXO1, BRD1, NCOA1 and NCOR2 with one local recurrence gaining three PIK3CA mutations. In contrast to DNA-level changes, we discovered recurrent outlier mRNA expression alterations were common--including outlier gains in TP63 (n=5 cases [42%]), NTRK3 [n=5 [42%]), NTRK2 (n=4 [33%]), PAX3 (n=4 [33%]), FGFR4 (n=3 [25%]) and TERT (n=3 [25%]). Recurrent losses involved ESR1 (n=5 [42%]), RELN (n=5 [42%]), SFRP4 (n=4 [33%]) and FOSB (n=4 [33%]). ESR1-depleted recurrences harbored shared transcriptional remodeling events including upregulation of PROM1 and other basal cancer markers. Taken together, this study defines acquired genomic changes in long-term, estrogen-deprived disease, highlights longitudinal RNA-profiling and identifies a common endocrine-resistant breast cancer subtype with basal-like transcriptional reprogramming.

cancer biology↗

bioRxiv2020

Susceptibility of tree shrew to SARS-CoV-2 infection

Since SARS-CoV-2 became a pandemic event in the world, it has not only caused huge economic losses, but also a serious threat to global public health. Many scientific questions about SARS-CoV-2 and COVID-19 were raised and urgently need to be answered, including the susceptibility of animals to SARS-CoV-2 infection. Here we tested whether tree shrew, an emerging experimental animal domesticated from wild animal, is susceptible to SARS-CoV-2 infection. No clinical signs were observed in SARS-CoV-2 inoculated tree shrews during this experiment except the increasing body temperature (above 39{degrees} C) particular in female animals during infection. Low levels of virus shedding and replication in tissues occurred in all three age groups, each of which showed his own characteristics. Histopathological examine revealed that pulmonary abnormalities were mild but the main changes although slight lesions were also observed in other tissues. In summary, tree shrew is not susceptible to SARS-CoV-2 infection and may not be a suitable animal for COVID-19 related researches.

microbiology↗

bioRxiv2020

Comparison of SARS-CoV-2 infections among 3 species of non-human primates

COVID-19, caused by SARS-CoV-2 infection, has recently been announced as a pandemic all over the world. Plenty of diagnostic, preventive and therapeutic knowledges have been enriched from clinical studies since December 2019. However, animal models, particularly non-human primate models, are urgently needed for critical questions that could not be answered in clinical patients, evaluations of anti-viral drugs and vaccines. In this study, two families of non-human primates, Old world monkeys (12 Macaca mulatta, 6 Macaca fascicularis) and New world monkeys (6 Callithrix jacchus), were experimentally inoculated with SARS-CoV-2. Clinical signs were recorded. Samples were collected for analysis of viral shedding, viremia and histopathological examination. Increased body temperature was observed in 100% (12/12) M. mulatta, 33.3% (2/6) M. fascicularis and none (0/6) of C. jacchus post inoculation of SARS-CoV-2. All of M. mulatta and M. fascicularis showed chest radiographic abnormality. Viral genomes were detected in nasal swabs, throat swabs, anal swabs and blood from all 3 species of monkeys. Viral shedding from upper respiratory samples reached the peak between day 6 and day 8 post inoculation. From necropsied M. mulatta and M. fascicularis, the tissues showing virus positive were mainly lung, weasand, bronchus and spleen. No viral genome was seen in any of tissues from 2 necropsied C. jacchus. Severe gross lesions and histopathological changes were observed in lung, heart and stomach of SARS-CoV-2 infected animals. In summary, we have established a NHP model for COVID-19, which could be used to evaluate drugs and vaccines, and investigate viral pathogenesis. M. mulatta is the most susceptible to SARS-CoV-2 infection, followed by M. fascicularis and C. jacchus. One Sentence SummaryM. mulatta is the most susceptible to SARS-CoV-2 infection as compared to M. fascicularis and C. jacchus.

microbiology↗

bioRxiv2020

Single cell transcriptomic heterogeneity in invasive ductal and lobular breast cancer cells

Invasive lobular breast carcinoma (ILC), one of the major breast cancer histological subtypes, exhibits unique clinical and molecular features compared to the other well-studied ductal cancer subtype (IDC). The pathognomonic feature of ILC is loss of E-cadherin, mainly caused by inactivating mutations within the CDH1 gene, but the extent of contribution of this genetic alteration to ILC-specific molecular characteristics remains largely understudied. To profile these features transcriptionally, we conducted single cell RNA sequencing on a panel of IDC and ILC cell lines, as well as an IDC cell line (T47D) with CRISPR-Cas9-mediated knock out (KO) of CDH1. Inspection of intra-cell line heterogeneity illustrated genetically and transcriptionally distinct subpopulations in multiple cell lines and highlighted rare populations of MCF7 cells highly expressing an apoptosis-related signature, positively correlated with a pre-adaptation signature to estrogen deprivation. Investigation of CDH1 KO-induced alterations showed transcriptomic membranous systems remodeling, elevated resemblance to ILCs in regulon activation, and suggests IRF1 as a potential mediator of reduced proliferation and increased cytokine-mediated immune-reactivity in ILCs.

cancer biology↗

bioRxiv2020

Measurement of the torque in braided DNAs using a thermodynamic Maxwell relation

Braided DNAs are significant structural intermediates in cellular processes, yet little has been experimentally demonstrated about their higher-order structure and twisting torques. We use magnetic tweezers to measure braid extensions at forces ranging from 0.3 to 8 piconewtons, and then apply a thermodynamic Maxwell relation to calculate the torque. Experimentally inferred torques in unbuckled braids take on values up to 76 pN{middle dot}nm, which depends on force, and inter-tether distance. As predicted using a statistical mechanical model, the twist modulus of the braids increases with catenation prior to buckling or formation of plectoneme, and is comparable to that of single DNA.

biophysics↗

bioRxiv2020

A hybrid spectral library combining DIA-MS data and a targeted virtual library substantially deepens the proteome coverage

Data-independent acquisition mass spectrometry (DIA-MS) is a rapidly evolving technique that enables relatively deep proteomic profiling with superior quantification reproducibility. DIA data mining predominantly relies on a spectral library of sufficient proteome coverage that, in most cases, is built on data-dependent acquisition-based analysis of the same sample. To expand the proteome coverage for a pre-determined protein family, we report herein on the construction of a hybrid spectral library that supplements a DIA experiment-derived library with a protein family-targeted virtual library predicted by deep learning. Leveraging this DIA hybrid library substantially deepens the coverage of three transmembrane protein families (G protein coupled receptors; ion channels; and transporters) in mouse brain tissues with increases in protein identification of 37-87%, and peptide identification of 58-161%. Moreover, of the 412 novel GPCR peptides exclusively identified with the DIA hybrid library strategy, 53.6% were validated as present in mouse brain tissues based on orthogonal experimental measurement.

bioinformatics↗

bioRxiv2019

Circulating bacterial DNA as a tool towards non-invasive biomarkers for colorectal cancer and adenoma

ObjectiveThe gut microbiota is closely associated with colorectal neoplasia. While most metagenomics studies utilized fecal samples, circulating microbial DNA in colorectal neoplasia patients remained unexplored. This study aimed to characterize microbial DNA in plasma samples and build a machine learning model for colorectal neoplasia early detection.\n\nDesignWe performed whole genome sequencing of plasma samples from 25 colorectal cancer (CRC) patients, 10 colorectal adenoma (CRA) patients and 22 healthy controls (HC). Microbial DNA was obtained by removing the host genome and relative abundance was measured by mapping reads into microbial genomes. Significant biomarker species were identified in the discovery cohort and built into a random forest model, which was tested in the validation cohort.\n\nResultsIn the discovery cohort, there were 127 significant species between CRC patients and HC. Based on the random forest model, 28 species were selected from the discovery cohort (AUC=0.944) and yielded an AUC of 1 in the validation cohort. Interestingly, relative abundance of most biomarker species in CRA patients were between CRC patients and HC with a trend towards CRC patients. Furthermore, pathway enrichment analysis also showed similar pattern where CRA patients had intermediate relative abundance of significant pathways compared to CRC patients and HC. Finally, species network analysis revealed that CRC and HC displayed distinct patterns of species association.\n\nConclusionsWe demonstrated characteristic alteration of circulating bacterial DNA in colorectal neoplasia patients. The predictive model accurately distinguished CRC and CRA from HC, suggesting the utility of circulating bacterial biomarkers as a non-invasive tool for colorectal neoplasia screening and early diagnosis.

cancer biology↗

bioRxiv2019

Inhibition of ADORA1 attenuates hepatic steatosis by gut microbiota-derived acetic acid from Astragalus polysaccharides

The authors have withdrawn this manuscript, because some data could not be replicated. During the process of subsequent revision of the manuscript, we separated the data into two parts. The majority data of the preprint draft, the anti-NAFLD effect of Astragalus polysaccharides (APS) in mice and the contribution of gut microbiota, were independently published in 2021 after substantial revision (doi: https://doi.org/10.1080/19490976.2021.1930874). However, the data on figure 6 (about ARs inhibitors DPCPX orally ameliorating NAFLD in mice, and Adora1 si-RNA data in vitro) could not be fully replicated in subsequent experiments. The most probable reason might be due to the batch differences of mice / cells, or other undetermined factors during experiment. Moreover, we have carried out in-depth studies on the exact role of hepatic A1R on NAFLD formation, which is opposite to our prior assumption. Therefore, we do not wish this work to be cited as reference for the project. We apologize for any inconvenience caused to our readers. Any questions can be addressed to first/corresponding author.

physiology↗

bioRxiv2019

Proteomic and Transcriptomic Profiling Identifies Mediators of Anchorage-Independent Growth and Roles of Inhibitor of Differentiation Proteins in Invasive Lobular Breast Cancer

BACKGROUNDInvasive lobular carcinoma (ILC) is a histological subtype of breast cancer with distinct molecular and clinical features from the more common subtype invasive lobular carcinoma (IDC). We have previously shown that human ILC cells lines have a remarkably unique ability to grow in ultra-low attachment (ULA) suspension cultures as compared to IDC cells, the mediators of which remain unknown. METHODSUsing flow cytometry and immunoblotting in human ILC and IDC cell lines, we measured levels of apoptosis and cell proliferation in attached (2D) and suspension (ULA) cultures. siRNA-mediated knockdown and pharmacological inhibitors were utilized to assess the effects of known regulators of anchorage-independence. Reverse Phase Protein Arrays and RNA-Sequencing were performed to identify novel proteomic and transcriptomic mediators of ULA growth in ILC cells. RESULTSWe show that human ILC cell lines exhibit enhanced anoikis resistance and cell proliferation in ULA cultures as compared to IDC cells. Transient restoration of E-cadherin did not impact the 2D or ULA growth of human ILC cell lines, while transient E-cadherin knockdown in IDC cells partially rescued their growth defect in ULA culture. Inhibition of the Rho/ROCK, p120-catenin or YAP/Hippo pathways previously implicated in anoikis resistance did not have a major effect on the ULA growth of ILC cells. Proteomic comparison of ILC and IDC cell lines identified unique induction of PI3K/Akt and p90-RSK pathways in ULA culture in ILC cells. Transcriptional profiling uncovered unique upregulation of the Inhibitors of Differentiation family transcription factors ID1 and ID3 in ILC ULA culture, the knockdown of which diminished anchorage-independent growth. We find that ID1 and ID3 expression is higher in human ILC tumors as compared to IDC and correlated with a worse disease-specific survival uniquely in the ILC cohort. CONCLUSIONOur comprehensive study of 2D and ULA growth in human ILC cell lines revealed anoikis resistance, cell proliferation and novel mediators of anchorage-independence and provides possible mechanistic insights and clinical implications for metastatic dissemination of ILC. High expression in human ILC tumors and association with clinical outcome implicate ID1 and ID3 as novel drivers and therapeutic targets for lobular breast cancer.

cancer biology↗