Resistance-associated Factors Research Articles

Abstract 540: Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development

Abstract Once cancer has metastasized, it is incurable because tumors evolve resistance to all systemically administered anti-cancer therapies. Resistance to therapy has classically been attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. Extracellular vesicles (EVs) are nano-sized (40-1000 nm) vesicles that are released by all cells into the extracellular space. EVs carry diverse cargo dependent on the cell of origin and have been studied as a source of novel cancer-specific liquid biopsy biomarkers and contributors to tumor progression and metastasis. However, the paracrine function of EVs in promoting therapy resistance is under-studied, and the impact of EVs released from therapy-resistant cancer cells on surrounding therapy-sensitive cancer cells is unknown. In this study, we develop an innovative microfluidic device, TREVCo, to enable simultaneous single-cell trap, EV detection, and co-culture modules to evaluate EVs-mediated transfer of cellular information between therapy-resistant cancer cells and adjacent cells, and to characterize EVs released from resistant cells and determine their impact on the resistance phenotype of adjacent therapy-sensitive cancer cells. We utilize resistant cells from multiple prostate (PCa) cell lines that have been selected for therapy resistance by culture with increasing dosages of cancer drugs such as cisplatin, docetaxel. We use the matched untreated therapy-sensitive parental cell lines as the sensitive cells. We hypothesize a novel model of therapy resistance in lethal PCa: EVs released from resistant cells contain pro-resistance factors that are transferred to adjacent sensitive cells upon EV uptake, thus directly enabling therapeutic resistance. We will assess the impact of resistant cell-EVs at a single cell level via a novel device, TREVCo, that enables comprehensive downstream analysis. This single-cell approach enables dissection of critical EV-associated therapy-resistant factors within the large heterogeneity of resistant PCa. Co-culture of a single resistant cell and a hundred adjacent sensitive cells mimicking intra-tumoral interactions is also critical to demonstrate evidence of resistant cell secreting factors inducing a pro-resistance phenotype. These studies will be the first-in-field to assess the impact of resistant PCa cell-derived EVs at single-cell resolution. An understanding of resistant cell-derived EVs and the impact of that EV cargo on adjacent sensitive cells will provide critical insight into the acquisition of therapy resistance in lethal PCa. The novel approach to apply resistant cell-derived EVs in co-culture assay with sensitive cells will enable the screening of transmittable resistance-associated factors and provides critical opportunities for accelerating precision medicine for advanced PCa patients. Citation Format: Chi-Ju Kim, Sarah Amend, Kenneth Pienta. Identification of therapy resistance mediating extracellular vesicles in single-cell level via a novel microfluidic co-culture device development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 540.

Leaf Bacteriome in Sugar Beet Shows Differential Response against Beet curly top virus during Resistant and Susceptible Interactions

Beet curly top virus (BCTV) significantly reduces sugar beet yield in semi-arid production areas. Genetic resistance to BCTV is limited; therefore, identification of additional resistance-associated factors is highly desired. Using 16S rRNA sequencing and BCTV resistant (R) genotypes (KDH13, KDH4-9) along with a susceptible (S) genotype (KDH19-17), we investigated leaf bacteriome changes during BCTV post inoculation (pi). At day 6 (~6-week-old plants), Cyanobacteria were predominant (~90%); whereas, at week 4 (~10-week-old plants) Firmicutes (11–66%), Bacteroidetes (17–26%), and Verrucomicrobia (12–29%) were predominant phyla and genotype dependent. Both Bacteroidetes and Verrucomicrobia, increased post infection only in the R lines. The bacterial genera Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the R lines. Linear discriminant analysis effect size (LEfSe) identified potential biomarkers in the R vs. S lines. Functional profiling revealed bacterial enrichment associated with the TCA cycle, polyisoprenoid, and L-methionine biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the R lines, and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the S line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents/biomarkers against BCTV.

Analysis of the Correlation Between Antibiotic Resistance Patterns and Virulence Determinants in Pathogenic Klebsiella pneumoniae Isolates from Egypt.

Klebsiella pneumoniae is responsible for a plethora of infections involving multiple body systems. This study investigated K. pneumoniae clinical isolates for virulence-associated characters and antibiotic resistance. First, antibiotic sensitivity was determined for 40 K. pneumoniae clinical isolates. Some virulence and resistance-associated factors were studied phenotypically and genotypically. Multiple resistance profiles were observed (multidrug resistant [MDR; 42.5%], extensive drug resistant [XDR; 35%], and pandrug resistant [PDR; 5%]). Moreover, CTX-M-1, TEM, qnrS, and qnrA genes were detected in 70%, 30%, 60%, and 30% of selected isolates, respectively, and 40% of tested isolates were extended-spectrum β-lactamases (ESBLs) producers. Interestingly, all ESBLs producers harbored class 1 integrase gene (IntI1), while 60% of ESBLs producers harbored both CTX-M-1 and TEM. All tested isolates were capsulated while 87.5% were biofilm producers. Fimbriae were detected in 90% of tested isolates (all were biofilm producers and type 3 fimbriae adhesion gene [mrkD] positive). Sequence analysis of OXA-48, qnrS, and IntI1 revealed 100% identity with published sequences, while sequencing of qnrA, OmpK-35, and iron regulatory protein gene (irp2) showed minor variations in the form of one or few single-nucleotide polymorphism. Altogether, the current study revealed that all MDR, XDR, and PDR K. pneumoniae isolates were multivirulent and all harbored 3-5 virulence genes and 2-9 antimicrobial resistance genes and exhibited 8 and 10 different virulence and antimicrobial resistance profiles, respectively. In this study, we also report a positive correlation between some virulence genes and antimicrobial resistance genes among K. pneumoniae tested isolates.

Comparative Proteomics and Secretomics Revealed Virulence and Antibiotic Resistance-Associated Factors in Vibrio parahaemolyticus Recovered From Commonly Consumed Aquatic Products.

Vibrio parahaemolyticus is a seafoodborne pathogen that can cause severe gastroenteritis and septicemia diseases in humans and even death. The emergence of multidrug-resistant V. parahaemolyticus leads to difficulties and rising costs of medical treatment. The bacterium of environmental origins containing no major virulence genes (tdh and trh) has been reported to be associated with infectious diarrhea disease as well. Identification of risk factors in V. parahaemolyticus is imperative for assuming food safety. In this study, we obtained secretomic and proteomic profiles of V. parahaemolyticus isolated from 12 species of commonly consumed aquatic products and identified candidate protein spots by using two-dimensional gel electrophoresis and liquid chromatography tandem mass spectrometry techniques. A total of 11 common and 28 differential extracellular proteins were found from distinct secretomic profiles, including eight virulence-associated proteins: outer membrane channel TolC, maltoporin, elongation factor Tu, enolase, transaldolase, flagellin C, polar flagellin B/D, and superoxide dismutase, as well as five antimicrobial and/or heavy metal resistance-associated ABC transporter proteins. Comparison of proteomic profiles derived from the 12 V. parahaemolyticus isolates also revealed five intracellular virulence-related proteins, including aldehyde-alcohol dehydrogenase, outer membrane protein A, alkyl hydroperoxide reductase C, phosphoenolpyruvate-protein phosphotransferase, and phosphoglycerate kinase. Additionally, our data indicated that aquatic product matrices significantly altered proteomic profiles of the V. parahaemolyticus isolates with a number of differentially expressed proteins identified. The results in this study meet the increasing need for novel diagnosis candidates of the leading seafoodborne pathogen worldwide.

PC-FACS

PC-FACS

Mapping the resistance-associated mobilome of a carbapenem-resistant Klebsiella pneumoniae strain reveals insights into factors shaping these regions and facilitates generation of a 'resistance-disarmed' model organism.

This study aims to investigate the landscape of the mobile genome, with a focus on antibiotic resistance-associated factors in carbapenem-resistant Klebsiella pneumoniae. The mobile genome of the completely sequenced K. pneumoniae HS11286 strain (an ST11, carbapenem-resistant, near-pan-resistant, clinical isolate) was annotated in fine detail. The identified mobile genetic elements were mapped to the genetic contexts of resistance genes. The blaKPC-2 gene and a 26 kb region containing 12 clustered antibiotic resistance genes and one biocide resistance gene were deleted, and the MICs were determined again to ensure that antibiotic resistance had been lost. HS11286 contains six plasmids, 49 ISs, nine transposons, two separate In2-related integron remnants, two integrative and conjugative elements (ICEs) and seven prophages. Sixteen plasmid-borne resistance genes were identified, 14 of which were found to be directly associated with Tn1721-, Tn3-, Tn5393-, In2-, ISCR2- and ISCR3-derived elements. IS26 appears to have actively moulded several of these genetic regions. The deletion of blaKPC-2, followed by the deletion of a 26 kb region containing 12 clustered antibiotic resistance genes, progressively decreased the spectrum and level of resistance exhibited by the resultant mutant strains. This study has reiterated the role of plasmids as bearers of the vast majority of resistance genes in this species and has provided valuable insights into the vital role played by ISs, transposons and integrons in shaping the resistance-coding regions in this important strain. The 'resistance-disarmed' K. pneumoniae ST11 strain generated in this study will offer a more benign and readily genetically modifiable model organism for future extensive functional studies.

Intrinsic breast cancer subtypes defined by estrogen receptor signalling—prognostic relevance of progesterone receptor loss

The majority of luminal type breast carcinomas are slowly growing tumors with an overall favorable prognosis. However, a proportion of cases (luminal B tumors) are characterized by coactivation of growth factor receptors or non-canonical ER signaling and a poorer clinical outcome. The aim of our study was to evaluate whether the expression of proteins that are part of the ER signaling network may be used to distinguish low-risk from high-risk luminal tumors. Unsupervised hierarchical clustering of a set of proteins either involved in estrogen receptor signaling or associated with resistance to endocrine therapy was performed in a series of 443 postmenopausal breast carcinomas. Using this approach, we were able to reproduce the established classification with two distinct groups of luminal (estrogen receptor positive) tumors, one group of HER2-associated tumors and a group of triple-negative tumors. However, neither proliferation nor the expression of one or more of the ER-co-factors or resistance-associated factors, but PR-expression was identified as the most important stratifier distinguishing between the two luminal groups. In fact, not only the four identified clusters were shown to be significantly associated with patient outcome, PR-expression alone or in combination with Ki–67-stains stratified ER-positive tumors into a low-risk and a high-risk group. Our data indicate that defining luminal B tumors by the presence of high-risk criteria (loss of PR-expression or increased proliferation) provides a robust and highly significant stratification of ER-positive breast carcinomas into luminal A and B.

Effect of hypoxia inducible factor-1α on multidrug resistance of gastric cancer cells and mechanism

Objective To investigate the expression of hypoxia inducible factor-1α (HIF-1α) in gastric cancer tissue and cell lines,and to study the effect of HIF-1α on multidrug resistance (MDR) of gastric cancer cells and mechanism.Methods The expression of HIF-1α in gastric cancer tissue,adjacent tissue and cell lines was detected by using Western blotting.HIF-1 α-siRNA was synthesized and tansfected into gastric cancer cell line OCUM-2MD3.Inhibition rate to tumor cells by chemotherapeutic agents was measured by using methyl thiazol tetrazolium (MTT) assay in vitro.The expression levels of multidrug resistance 1 (MDR1)/permeability glycoprotein (P-gp),glutathione S-transferase-π (GST-π),B lymphocytes/leukemia-2 (bcl-2) and bax were determined by using quantitative reverse transcription-polymerase chain reaction (qPCR) and Western blotting.Results Overexpression of HIF-1α was detected in gastric cancer tissue and cell lines:mRNA levels were (0.853 ±0.182) and (0.497 ±0.088),and protein levels were (0.952 ±0.198) and 0.325 ±0.052) (all P ＜0.01).The expression of HIF-1α was inhibited by HIF-1α-siRNA with the optimal dose being 80 nmol/L and optimal time being 48 h:mRNA from (0.747 ±0.087) to (0.177 ±0.043),and protein from (0.622 ±0.089) to (0.211 ±0.032) (all P＜0.05).Inhibition rate to tumor cells by chemotherapeutic agents was increased after HIF-1α-siRNA tansfected into gastric cancer cells (all P ＜ 0.05).The expression levels of MDR1/P-gp,Survivin and bcl-2 mRNA were lower (0.44 ±0.06,0.46 ±0.07,0.37 ±0.08 respectively) in cells after HIF-1α-siRNA tansfection than before transfection (0.90±0.11,1.04 ±0.10,1.01 ±0.19 respectively) (all P＜0.05),but the expression of bax was higher after transfection than before transfection (1.91 ±0.09 vs.1.91 ±0.09) (P ＜0.05).The protein expression levels of P-gp,GST-r,and bcl-2 were reduced from (1.58 ± 0.19),(1.37 ± 0.17) and (1.97 ± 0.22) to (0.82 ± 0.12),(0.51 ± 0.06) and (0.81 ± 0.09) respectively,and the protein expression level of bax was increased from (0.51 ± 0.07) to (1.03 ± 0.01) (all P ＜0.05).Conclusion Close relation exsists between HIF-1α and MDR of gastric cancer,and inhibiting HIF-1 α can reverse MDR of gastric cancer cells by regulating the expressions of multidrug resistance associated factors. Key words: Gastric carcinomar; Hypoxia inducible factor-1 α; Multidrug resistance

Relationship between expressions of multidrug resistance associated factors and chemosensitivity to docetaxel in lymph node metastases of gastric carcinoma

Objective To investigate the relationship between the expression of multidrug resist-ante (MDR) associated factors and ehemosensitivity of docetaxel (TXT) in lymph node metastases of gas-trie carcinoma. Methods The chemosenisitivity to TXT was measured by MTT assay, and the expression of P-gp, GST-π, p53 and Survivin was detected immunohistochemically in 54 paired primary tumor (PT) and LNMs of gastric carcinoma. Results There was statistically negative correlation between inhibitory rate and the expression of P-gp, GST-π in PT (both P < 0. 05 ), and in LNMs only Survivin had a negative correlation with inhibitory rate for TXT (P < 0.05 ). The expression of P-gp and GST-π was higher in LNMs than in PT (both P < 0. 05 ) ; and positive correlation of P-gp, p53 was found between PT and LNMs (r = 0. 3424, 0. 7123, both P < 0. 05 ). Conclusion The main factors influencing chemosensitivity to TXT were different between PT and LNMs. Key words: Gastric neoplasms; Metastases ; Docetaxel; Chemosensitivity

The Phosphorylation of Ser318 of Insulin Receptor Substrate 1 Is Not per se Inhibitory in Skeletal Muscle Cells but Is Necessary to Trigger the Attenuation of the Insulin-stimulated Signal

The Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS) is one key mechanism to stimulate and/or attenuate insulin signal transduction. Using a phospho-specific polyclonal antibody directed against phosphorylated Ser(318) of IRS-1, we found a rapid and transient insulin-stimulated phosphorylation of Ser(318) in human and rodent skeletal muscle cell models and in muscle tissue of insulin-treated mice. None of the investigated insulin resistance-associated factors (e.g. high glucose, tumor necrosis factor-alpha, adrenaline) stimulated the phosphorylation of Ser(318). Studying the function of this phosphorylation, we found that replacing Ser(318) by alanine completely prevented both the attenuation of insulin-stimulated Akt/protein kinase B Ser(473) phosphorylation and glucose uptake after 60 min of insulin stimulation. Unexpectedly, after acute insulin stimulation, we observed that phosphorylation of Ser(318) is not inhibitory but rather enhances insulin signal transduction because introduction of Ala(318) led to a reduction of the insulin-stimulated Akt/protein kinase B phosphorylation. Furthermore, replacing Ser(318) by glutamate, i.e. mimicking phosphorylation, improved glucose uptake after acute insulin stimulation. These data suggest that phosphorylation of Ser(318) is not per se inhibitory but is necessary to trigger the attenuation of the insulin-stimulated signal in skeletal muscle cells. Investigating the molecular mechanism of insulin-stimulated Ser(318) phosphorylation, we found that phosphatidylinositol 3-kinase-mediated activation of atypical protein kinase C-zeta and recruitment of protein kinase C-zeta to IRS-1 was responsible for this phosphorylation. We conclude that Ser(318) phosphorylation of IRS-1 is an early physiological event in insulin-stimulated signal transduction, which attenuates the continuing action of insulin.

Intrinsic expression of drug resistance-associated factors in meningiomas.

Meningiomas, commonly benign tumors, rarely display aggressive behavior by recurrences and invasion. In addition to surgery, irradiation is beneficial for recurrent, atypical, and malignant meningiomas. The role of chemotherapy, however, remains controversial, although there is evidence that meningiomas respond well to adjuvant chemotherapy. A major obstacle in chemotherapy remains drug resistance with reduced cellular drug accumulation through membrane efflux pumps, drug detoxification, and alterations in drug target specificity. In 84 classic, atypical, and malignant meningiomas, the immunohistochemical expression profile of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), metallothionein, and topoisomerase IIalpha were studied. All types of meningiomas showed constant expression of P-gp, LRP, MRP, and topoisomerase IIalpha; metallothionein was found in 67% of the tumors, especially in atypical and malignant meningiomas. Furthermore, metallothionein. P-gp, LRP, and topoisomerase IIalpha were strongly expressed by normal and neoplastic vessels, which may confer to impaired penetration of therapeutic agents through the blood-brain and blood-tumor barrier. Neither recurrent nor previously irradiated meningiomas revealed any significant difference to primary tumors. These intrinsic drug resistances indicate that successful chemotherapy may require additional inhibition of these factors to be a promising approach in the management of meningiomas.

Drug resistance-associated factors in primary and secondary glioblastomas and their precursor tumors.

Malignant gliomas are largely resistant to current chemotherapeutic strategies often displaying a multidrug-resistant phenotype. Mechanisms involved in drug resistance are reduced cellular drug accumulation through membrane efflux pumps, drug detoxification as well as alterations in drug target specificity. In 27 primary and 17 secondary glioblastomas and their astrocytic precursor tumors, we studied the immunohistochemical expression profile of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), metallothionein, and topoisomerase II alpha. Glial tumor cells in all glioblastomas showed constant up-regulation of LRP, MRP, and topoisomerase II alpha. P-gp was found in 90% of the primary and 60% of the secondary glioblastomas. In precursor tumors, these drug resistance-related factors were expressed in varying proportions. Metallothionein, also found in normal and activated astrocytes, was retained in all neoplastic phenotypes. Furthermore, metallothionein, P-gp, LRP, and topoisomerase II alpha were strongly expressed by normal and neoplastic vessels which may confer to impaired penetration of therapeutic agents through the blood-brain and blood-tumor barrier. However, the expression profiles of drug resistance-related proteins neither differed between primary and secondary glioblastomas nor revealed any correlation to precursor or recurrent tumors. Nevertheless, inhibition of these factors may be promising approaches to the management of malignant gliomas.

Resistance-associated factors in human small-cell lung-carcinoma GLC 4 sub-lines with increasing adriamycin resistance : Versantvoort CHM, Withoff S, Broxterman HJ, Kuiper CM, Scheper RJ, Mulder NH et al. Department of Medical Oncology, Free University Hospital, de Boelelaan 1117, 1081 HV Amsterdam. Int J Cancer 1995;61:375–380

Resistance-associated factors in human small-cell lung-carcinoma GLC 4 sub-lines with increasing adriamycin resistance : Versantvoort CHM, Withoff S, Broxterman HJ, Kuiper CM, Scheper RJ, Mulder NH et al. Department of Medical Oncology, Free University Hospital, de Boelelaan 1117, 1081 HV Amsterdam. Int J Cancer 1995;61:375–380