Expression Of Epithelial-mesenchymal Transition Markers Research Articles

Shear Stress Drives the Cleavage Activation of Protease-Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells.

When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment-induced anoikis and fluidic shear stress (SS)-mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS-specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease-activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos-related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3to cleave the N-terminal inhibitory domain of PAR2within 2h. As a G protein-coupled receptor, PAR2further activates the Gαi protein to turn on the Src-ERK/p38/JNK-FRA1/cJUN axis to promote the expression of epithelial-mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2,and FOSL1in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2may serve as an SS-specific mechanosensor cleavable by PRSS3in circulation, which provides new insights for targeting metastasis-initiating CTCs.

Vitamin D deficiency contributes to the diabetic kidney disease progression via increase ZEB1/ZEB2 expressions

BackgroundDiabetic kidney disease (DKD) remains one of the main causes of end-stage renal disease (ESRD) and mortality in diabetic patients worldwide. Vitamin D deficiency (VitDD) is one of the main consequences of different chronic kidney disease (CKD) types and is associated with rapid progression to ESRD. Nevertheless, the mechanisms that lead to this process are poorly understood. This study aimed to characterize a model of diabetic nephropathy progression in VitDD and the epithelial-mesenchymal-transition (EMT) role in these processes.MethodsWistar Hannover rats received a diet with or without VitD before type 1 diabetes (T1D) induction. After this procedure, the rats were accompanied for 12 and 24 weeks after T1D induction and the renal function, structure, cell transdifferentiating markers and zinc finger e-box binding homeobox 1/2 (ZEB1/ZEB2) contribution to kidney damage were evaluated during the DKD progression.ResultsThe results showed an increase in glomerular tuft, mesangial and interstitial relative areas and renal function impairment in VitD-deficient diabetic rats compared to diabetic rats that received a VitD-containing diet. These alterations can be associated with increased expression of EMT markers, ZEB1 gene expression, ZEB2 protein expression and TGF-β1 urinary excretion. Decreased miR-200b expression, an important post-transcriptional regulator of ZEB1 and ZEB2 was also observed.ConclusionOur data demonstrated that VitD deficiency contributes to the rapid development and progression of DKD in diabetic rats induced by increase ZEB1/ZEB2 expressions and miR-200b downregulation.

Recognition Imaging of Trace E-cadherins on the Bladder Cancer Cells Surface during Epithelial-Mesenchymal Transition by Force-Distance Curve-Based AFM.

Tumor-associated epithelial-mesenchymal transition (EMT) contains a set of transitional cellular states usually judged by the EMT marker expression. E-cadherin is a down-regulated EMT epithelial marker, and the detection of E-cadherin is challenging on cancer cell surfaces in the middle and late stages of EMT. Here, the trace E-cadherins on the living bladder cancer T24 cell surface during EMT were investigated with force-distance curve-based atomic force microscopy. The results confirmed that T24 cells are still in an intermediate state and can be transferred into the mesenchymal phenotype by long-term TGF-β1 induction. During EMT, E-cadherins on the T24 cell surface gradually decreased and rarely clustered. E-cadherin is not completely missing, even at the end of EMT, but is too sparse to cluster. This work provides us with a visual understanding of the expression and distribution of trace markers during EMT and a deep comprehension of the indispensable significance of E-cadherin in cancer cells.

Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia

ObjectiveJiedu Recipe (JR), a Chinese herbal remedy, has been shown to prolong overall survival time and decrease recurrence and metastasis rates in patients with hepatocellular carcinoma (HCC). This work investigated the mechanism of JR in HCC treatment. MethodsThe chemical constituents of JR were detected using liquid chromatography-mass spectrometry. The potential anti-HCC mechanism of JR was screened using network pharmacology and messenger ribonucleic acid (mRNA) microarray chip assay, followed by experimental validation in human HCC cells (SMMC-7721 and Huh7) in vitro and a nude mouse subcutaneous transplantation model of HCC in vivo. HCC cell characteristics of proliferation, migration and invasion under hypoxic setting were investigated using thiazolyl blue tetrazolium bromide, wound healing and Transwell assays, respectively. Image-iT™ Hypoxia Reagent was added to reveal hypoxic conditions. Stem cell sphere formation assay was used to detect the stemness. Epithelial-mesenchymal transition (EMT) markers like E-cadherin, vimentin and α-smooth muscle actin, and pluripotent transcription factors including nanog homeobox, octamer-binding transcription factor 4, and sex-determining region Y box protein 2 were analyzed using Western blotting and real-time polymerase chain reaction. Western blot was performed to ascertain the anti-HCC effect of JR under hypoxia involving the Wnt/β-catenin pathway. ResultsAccording to network pharmacology and mRNA microarray chip analysis, JR may potentially act on hypoxia and inhibit the Wnt/β-catenin pathway. In vitro and in vivo experiments showed that JR significantly decreased hypoxia, and suppressed HCC cell features of proliferation, migration and invasion; furthermore, the hypoxia-induced increases in EMT and stemness marker expression in HCC cells were inhibited by JR. Results based on the co-administration of JR and an agonist (LiCl) or inhibitor (IWR-1-endo) verified that JR suppressed HCC cancer stem-like properties under hypoxia by blocking the Wnt/β-catenin pathway. ConclusionJR exerts potent anti-HCC effects by inhibiting cancer stemness via abating the Wnt/β-catenin pathway under hypoxic conditions.Please cite this article as: Guo BJ, Ruan Y, Wang YJ, Xiao CL, Zhong ZP, Cheng BB, Du J, Li B, Gu W, Yin ZF. Jiedu Recipe, a compound Chinese herbal medicine, inhibits cancer stemness in hepatocellular carcinoma via Wnt/β-catenin pathway under hypoxia. J Integr Med. 2023; 21(5): 474–486.

P18INK4C and BRCA1 inhibit follicular cell proliferation and dedifferentiation in thyroid cancer

ABSTRACT Only 3% of thyroid cancers are medullary thyroid carcinomas (MTCs), the rest are follicular epithelial cell derived non-MTCs (NMTCs). A dysfunctional INK4-CDK4-RB pathway is detected in most of NMTCs. DNA repair defects and genome instability are associated with NMTC dedifferentiation and aggressiveness. Whether inactivation of the INK4-CDK4-RB pathway induces NMTCs and how differentiation of NMTC cells is controlled remain elusive. In this study, we generated p18Ink4c and Brca1 singly and doubly deficient mice as well as p16Ink4a and Brca1 singly and doubly deficient mice. By using these mice and human thyroid carcinoma cell lines, we discovered that loss of p18Ink4c, not p16Ink4a, in mice stimulated follicular cell proliferation and induced NMTCs. Depletion of Brca1 alone or both p16Ink4a and Brca1 did not induce thyroid tumor. Depletion of Brca1 in p18Ink4c null mice results in poorly differentiated and aggressive NMTCs with epithelial-mesenchymal transition (EMT) features and enhanced DNA damage. Knockdown of BRCA1 in thyroid carcinoma cells activated EMT and promoted tumorigenesis whereas overexpression of BRCA1 inhibited EMT. BRCA1 and EMT marker expression were inversely related in human thyroid cancers. Our finding, for the first time, demonstrates that inactivation of INK4-CDK4-RB pathway induces NMTCs and that Brca1 deficiency promotes dedifferentiation of NMTC cells. These results suggest that BRCA1 and p18INK4C collaboratively suppress thyroid tumorigenesis and progression and CDK4 inhibitors will be effective for treatment of INK4-inactivated or cyclin D-overexpressed thyroid carcinomas.

Acute myeloid leukemia (AML)-derived mesenchymal stem cells induce chemoresistance and epithelial-mesenchymal transition-like program in AML through IL-6/JAK2/STAT3 signaling.

Acute myeloid leukemia (AML) has a high rate of treatment failure due to increased prevalence of therapy resistance. Mesenchymal stem cells (MSCs) in the leukemia microenvironment contribute to chemoresistance in AML, but the specific mechanism remains unclear. The critical role of the epithelial-mesenchymal transition (EMT)-like profile in AML chemoresistance has been gradually recognized. However, there is no research to suggest that the AML-derived bone marrow mesenchymal stem cells (AML-MSCs) induce the EMT program in AML thus far. We isolated AML-MSCs and cocultured them with AML cells. We found that AML-MSCs induced a significant mesenchymal-like morphology in drug-resistant AML cells, but it was scarce in parental AML cells. The AML-MSCs promoted growth of AML cells in the presence or absence of chemotherapeutics in vitro and in vivo. Acute myeloid leukemia MSCs also induced EMT marker expression in AML cells, especially in chemoresistant AML cells. Mechanistically, AML-MSCs secreted abundant interleukin-6 (IL-6) and upregulated IL-6 expression in AML cells. Acute myeloid leukemia cells upregulated IL-6 expression in AML-MSCs in turn. Meanwhile, AML-MSCs activated the JAK2/STAT3 pathway in AML cells. Two JAK/STAT pathway inhibitors counteracted the AML-MSCs induced morphology change and EMT marker expression in AML cells. In conclusion, AML-MSCs not only promote the emergence of chemoresistance but also enhance it once AML acquires chemoresistance. AML-MSCs induce EMT-like features in AML cells; this phenotypic change could be related to chemoresistance progression. AML-MSCs induce the EMT-like program in AML cells through IL-6/JAK2/STAT3 signaling, which provides a therapeutic target to reverse chemoresistance in AML.

The CCL2-CCR2 Axis Contributes to Migration of Cabazitaxel-resistant Prostate Cancer Cells.

Developing resistance to cabazitaxel is a major challenge in patients with docetaxel- and castration-resistant prostate cancer (CRPC) since it is frequently administered as a last resort. We have previously reported that CCL2 induces resistance to the antiproliferative effect of cabazitaxel in DU145-TxR/CxR prostate cancer cell lines. However, how CCL2 induces resistance to the antimigration effect of cabazitaxel remains unclear. We established a cabazitaxel-resistant cell line, DU145-TxR/CxR, from a previously established paclitaxel-resistant cell line, DU145-TxR, which was confirmed to show docetaxel resistance. We performed migration assay and analyzed the expression of epithelial-mesenchymal transition markers using DU145-TxR/CxR with or without CCL2 silencing with small interfering RNA (siRNA) transfection. Cabazitaxel inhibited the migration of DU145 cells through the inactivation of STAT3. A CCR2 (a specific receptor of CCL2) antagonist suppressed the migration of DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. Western blotting revealed that the CCR2 antagonist inhibited STAT3 phosphorylation in DU145-TxR and DU145-TxR/CxR cells under cabazitaxel treatment. CCL2 silencing with siRNA in DU145-TxR and DU145-TxR/CxR cells decreased migration through STAT3 and p38 inactivation. Furthermore, CCL2 activated AKT, and CCR2 antagonist inhibited AKT phosphorylation in DU145-TxR and DU145-TxR/CxR cells with recovery of sensitivity to cabazitaxel under cabazitaxel treatment. The CCL2-CCR2 axis is a key contributor to resistance to the antimigration effect of cabazitaxel in prostate cancer cells. CCL2-CCR2 axis inhibition may be a potential therapeutic target against chemoresistant CRPC in combination with cabazitaxel.

Soluble dipeptidyl peptidase-4 induces epithelial-mesenchymal transition through tumor growth factor-β receptor.

Kidney fibrosis is the final manifestation of chronic kidney disease, a condition mainly caused by diabetic nephropathy. Persistent tissue damage leads to chronic inflammation and excessive deposition of extracellular matrix (ECM) proteins. Epithelial-mesenchymal transition (EMT) is involved in a variety of tissue fibrosis and is a process during which epithelial cells transform into mesenchymal-like cells and lose their epithelial functionality and characteristics Dipeptidyl peptidase-4 (DPP4) is widely expressed in tissues, especially those of the kidney and small intestine. DPP4 exists in two forms: a plasma membrane-bound and a soluble form. Serum-soluble DPP4 (sDPP4) levels are altered in many pathophysiological conditions. Elevated circulating sDPP4 is correlated with metabolic syndrome. Because the role of sDPP4 in EMT remains unclear, we examined the effect of sDPP4 on renal epithelial cells. The influences of sDPP4 on renal epithelial cells were demonstrated by measuring the expression of EMT markers and ECM proteins. sDPP4 upregulated the EMT markers ACTA2 and COL1A1 and increased total collagen content. sDPP4 activated SMAD signaling in renal epithelial cells. Using genetic and pharmacological methods to target TGFBR, we observed that sDPP4 activated SMAD signaling through TGFBR in epithelial cells, whereas genetic ablation and treatment with TGFBR antagonist prevented SMAD signaling and EMT. Linagliptin, a clinically available DPP4 inhibitor, abrogated sDPP4-induced EMT. This study indicated that sDPP4/TGFBR/SMAD axis leads to EMT in renal epithelial cells. Elevated circulating sDPP4 levels may contribute to mediators that induce renal fibrosis.

The Role of ROR1 in Chemoresistance and EMT in Endometrial Cancer Cells.

Background and Objectives: Receptor tyrosine kinase-like orphan receptor type 1 (ROR1) plays a critical role in embryogenesis and is overexpressed in many malignant cells. These characteristics allow ROR1 to be a potential new target for cancer treatment. The aim of this study was to investigate the role of ROR1 through in vitro experiments in endometrial cancer cell lines. Materials and Methods: ROR1 expression was identified in endometrial cancer cell lines using Western blot and RT-qPCR. The effects of ROR1 on cell proliferation, invasion, migration, and epithelial-mesenchymal transition (EMT) markers were analyzed in two endometrial cancer cell lines (HEC-1 and SNU-539) using either ROR1 silencing or overexpression. Additionally, chemoresistance was examined by identifying MDR1 expression and IC50 level of paclitaxel. Results: The ROR1 protein and mRNA were highly expressed in SNU-539 and HEC-1 cells. High ROR1 expression resulted in a significant increase in cell proliferation, migration, and invasion. It also resulted in a change of EMT markers expression, a decrease in E-cadherin expression, and an increase in Snail expression. Moreover, cells with ROR1 overexpression had a higher IC50 of paclitaxel and significantly increased MDR1 expression. Conclusions: These in vitro experiments showed that ROR1 is responsible for EMT and chemoresistance in endometrial cancer cell lines. Targeting ROR1 can inhibit cancer metastasis and may be a potential treatment method for patients with endometrial cancer who exhibit chemoresistance.

Epithelial to mesenchymal transition in mammary gland tissue fibrosis and insights into drug therapeutics.

The epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process in which epithelial cells lose their epithelial properties and gain mesenchymal characteristics. The process of EMT has been shown to mediate mammary gland fibrosis. Understanding how mesenchymal cells emerge from an epithelial default state will aid in unravelling the mechanisms that control fibrosis and, ultimately, in identifying therapeutic targets to alleviate fibrosis. The effects of EGF and high glucose (HG) on EMT in mammary epithelial cells, MCF10A and GMECs, as well as their pathogenic role, were studied. In-silico analysis was used to find interacting partners and protein-chemical/drug molecule interactions. On treatment with EGF and/or HG, qPCR analysis showed a significant increase in the gene expression of EMT markers and downstream signalling genes. The expression of these genes was reduced on treatment with EGF+HG combination in both cell lines. The protein expression of COL1A1 increased as compared to the control in cells treated with EGF or HG alone, but when the cells were treated with EGF and HG together, the protein expression of COL1A1 decreased. ROS levels and cell death increased in cells treated with EGF and HG alone, whereas cells treated with EGF and HG together showed a decrease in ROS production and apoptosis. In-silico analysis of protein-protein interactions suggest the possible role of MAPK1, actin alpha 2 (ACTA2), COL1A1, and NFκB1 in regulating TGFβ1, ubiquitin C (UBC), specificity protein 1 (SP1) and E1A binding protein P300 (EP300). Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment suggests advanced glycation end products-receptor for advanced glycation end products (AGE-RAGE) signalling pathway, relaxin signalling pathway and extra cellular matrix (ECM) receptor interactions underlying fibrosis mechanism. This study demonstrates that EGF and HG induce EMT in mammary epithelial cells and may also have a role in fibrosis.

Analysis of the protumoral mechanisms of F.nucleatumon growth, epithelial-mesenchymal transition and the expression of immunosuppressive markers in cell line of OSCC

Abstract Background: Oral squamous cell carcinoma (OSCC) is the most common manifestation of oral cancer. It has been proposed that periodontal pathogens contribute to OSCC progression, and preliminary data from our laboratory has identified Fusobacterium nucleatum (F.n) and Epithelial Mesenchymal Transition (EMT) markers in the secretome of biopsies from patients with OSCC in comparison with healthy biopsies from the oral cavity. However, the mechanisms modulated by the tumoral bacteriome in OSCC remains not fully understood. Methods: Oral cancer cells (HSC3) were infected with F.n using a MOI of 100 to evaluate the effect of the bacteria on tumor growth, expression of EMT markers and immunomodulatory properties. Results: After infection, we confirmed the presence of bacteria inside and surrounding the cancer cells by confocal microscopy. Then, a significant increment in the size of F.n-infected tumor spheroids was found at 3-, 6- and 10-days post-infection. Related to EMT markers, MMP-9 transcripts were significantly elevated in infected cells, whereas E-cadherin transcripts were significantly downregulated post infection. In addition, the Human XL Oncology array kit revealed that several cancer proteins such as MMP-3, GM-CSF, FOXO1/FKHR, Cathepsina D-S, Amphiregulin, SerpinB5/Maspin and EGFR were upregulated and others like ICAM-1, DLL-1 and ERB2 were downregulated after F.n-infection. Finally, the expression of Galectin-9 measured by flow cytometry was significantly increased in infected cells than control cancer cells. Conclusions: The periodontal bacterium F.n promotes tumor progression of OSCC through increased tumor growth, acquisition of ETM-associated markers and Galectin-9 upregulation. Fondecyt Regular Project 1211480

Prophylaxis of posterior capsule opacification through autophagy activation with indomethacin-eluting intraocular lens.

Posterior capsule opacification (PCO) is the most common long-term postoperative complication of cataract surgery, leading to secondary vision loss. Optimized intraocular lens (IOL) structure and appropriate pharmacological intervention, which provides physical barriers and biological inhibition, respectively, can block the migration, proliferation, and epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) for PCO prophylaxis. Herein, a novel indomethacin-eluting IOL (INDOM-IOL) with an optimized sharper edge and a sustained drug release behavior was developed for PCO prevention. Indomethacin (INDOM), an ophthalmic non-steroidal anti-inflammatory drug (NSAID) used for postoperative ocular inflammation, was demonstrated to not only be able to suppress cell migration and down-regulate the expression of cyclooxygenase-2 (COX-2) and EMT markers, including alpha-smooth muscle actin (α-SMA) and cyclin D1, but also promote the autophagy activation in LECs. Additionally, autophagy was also verified to be a potential therapeutic target for the down-regulation of EMT in LECs. The novel IOL, serving as a drug delivery platform, could carry an adjustable dose of hydrophobic indomethacin with sustained drug release ability for more than 28 days. In the rabbit PCO model, the indomethacin-eluting IOL showed excellent anti-inflammatory and anti-PCO effects. In summary, indomethacin is an effective pharmacological intervention in PCO prophylaxis, and the novel IOL we developed prevented PCO in vivo under its sustained indomethacin release property, which provided a promising approach for PCO prophylaxis in clinical application.

The Immunohistochemical Expression of Epithelial-Mesenchymal Transition Markers in Precancerous Lesions and Cervical Cancer.

In the epithelial-mesenchymal transition (EMT) process, cells lose their epithelial phenotype and gain mesenchymal features. This phenomenon was observed in the metastatic phase of neoplastic diseases, e.g., cervical cancer. There are specific markers that are expressed in the EMT. The aim of this study was to determine the localization of and associations between the immunohistochemical (IHC) expression of TWIST, SNAIL, and SLUG proteins in precancerous lesions and cervical cancer. The IHC analysis disclosed higher expressions of EMT markers in precancerous lesions and cervical cancer than in the control group. Moreover, stronger expression of TWIST, SNAIL, and SLUG was observed in cervical intraepithelial neoplasia grade 3 (CIN3) vs. CIN1, CIN3 vs. CIN2, and CIN2 vs. CIN1 cases (p < 0.05). In cervical cancer, IHC reactions demonstrated differences in TWIST, SNAIL, and SLUG expression in grade 1 (G1) vs. grade 2 (G2) (p < 0.0011; p < 0.0017; p < 0.0001, respectively) and in G1 vs. grade 3 (G3) (p < 0.0029; p < 0.0005; p < 0.0001, respectively). The results of our study clearly showed that existing differences in the expression of the tested markers in precancerous vs. cancerous lesions may be utilized in the diagnosis of cervical cancer. Further studies on bigger populations, as well as in comparison with well-known markers, may improve our outcomes.

Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.

Never in mitosis gene A-related kinase-8 promotes proliferation, migration, invasion, and stemness of breast cancer cells via β-catenin signalling activation

Never in mitosis gene A (NIMA)-related kinase-8 (NEK8) is involved in cell cycle progression, cytoskeleton development, and DNA damage repair. However, its role in breast cancer has not yet been explored. To investigate this, NEK8 was knocked down in MDA-MB-231, BT549, and HCC38 breast cancer cell lines. We observed a decrease in cell proliferation and colony formation owing to regulation of the G1/S and G2/M transitions. Furthermore, the expression of several cell cycle regulatory proteins was altered, including that of cyclin D1, cyclin B1, CDK4, CDK2, and surviving. NEK8 knockdown impaired cell migration and invasion as well as reduced the expression of epithelial-mesenchymal transition markers. Regarding stem-cell characteristics, NEK8 knockdown decreased the tumour sphere formation, aldehyde dehydrogenase activity, and stem-cell marker expression, including that of CD44, Sox2, Oct4a, and Nanog. Further analysis revealed that NEK8 interacts with β-catenin. Also, NEK8 knockdown promoted β-catenin degradation. NEK8-silenced MDA-MB-231 cells inhibited xenograft tumour growth, metastasis, and tumour initiation in vivo. Using the Oncomine and TNMplot public databases, we found a significant correlation between NEK8 overexpression and poor clinical outcomes in breast cancer patients. Thus, NEK8 may be a crucial regulator of breast cancer progression and a potential therapeutic target.

Epithelial-mesenchymal Transition Factor and Its Association With Mammographic Density and Breast Cancer Prognosis.

The tumor microenvironment influences tumor progression, invasion, and metastasis. This study determined the expression levels of epithelial-mesenchymal transition (EMT) factors according to zone and their correlation with mammographic breast density and investigated the prognostic value of EMT factors. The clinical and pathological data of invasive carcinoma and ductal carcinoma in situ were reviewed. Primary breast tissue samples were evaluated using immunohistochemistry (IHC) staining of the EMT-associated markers, including α-SMA, vimentin, MMP-9, and CD34. The expression levels were analyzed in three areas: the tumor center, interface, and distal zones. EMT factors were correlated with mammographic breast density and oncologic outcomes. An overall EMT phenotype conversion from positive to negative was seen in 55.7% of α-SMA- and 34.4% of MMP-9-positive cells between the tumor center and interface zones, which was significantly different (p<0.05). Most changes in EMT expression from the center to the distal zone were from positive to negative, but 23.0% of CD34-expressing cells showed negative to positive conversion. The proportion of α-SMA, vimentin, and MMP-9 expression was higher in the non-dense breast group compared to the dense breast group in the interface and distal zones (p<0.05). CD34 expression in the distal zone was an independent favorable prognostic factor for disease-free survival (p=0.039). The differential expression of EMT markers in each zone suggests heterogeneous cancer cell populations within each zone of breast cancer. EMT factor expression can also interplay between breast density stroma and geographical tumor zone.

Prognostic significance of extranodal extension of nodal metastasis in adenocarcinoma of the ampulla of Vater

Although nodal metastasis (NM) is an important prognostic factor of ampullary adenocarcinoma, the prognostic implication of extranodal extension (ENE) is not well characterized. NM with ENE status was investigated in 279 surgically resected ampullary adenocarcinoma patients and compared with other clinicopathologic factors, including overall survival (OS) and recurrence-free survival (RFS). Expression of epithelial-mesenchymal transition (EMT) markers, including E-cadherin, Twist, and Snail, was assessed in a subset of the cohort. NM was observed in 94 cases (33.7%), of which ENE was observed in 32 cases (34%). NM with ENE was more frequently associated with tumors with poor differentiation than NM without ENE (P=.017). The 5-year OS and RFS rates of patients with NM and ENE was significantly worse (13.0% and 6.3%) than those with NM without ENE (37.7% and 21.4%) and those without NM (57.6% and 50.2%, respectively; P<.001). When pN category was matched, the OS and RFS was worse in patients with ENE than in those without ENE (P<.05). Moreover, the expression of E-cadherin and Twist was significantly different between NM areas with and without ENE (all, P<.001). Since ENE was associated with poorly differentiated ampullary adenocarcinomas and showed different expression of EMT markers, EMT could be a possible mechanism of ENE. Ampullary adenocarcinoma patients with ENE had worse OS and RFS than those without ENE. Therefore, evaluation of ENE can provide additional survival information for patients with surgically resected ampullary carcinoma.

Effective inhibition of breast cancer stem cell properties by quercetin-loaded solid lipid nanoparticles via reduction of Smad2/Smad3 phosphorylation and β-catenin signaling pathway in triple-negative breast cancer

BackgroundThe presence of cancer stem cells (CSCs) is a major cause of resistance to cancer therapy and recurrence. Triple-negative breast cancer (TNBC) is a subtype that responds poorly to therapy, making it a significant global health issue. Quercetin (QC) has been shown to affect CSC viability, but its low bioavailability limits its clinical use. This study aims to increase the effectiveness of QC in inhibiting CSC generation by using solid lipid nanoparticles (SLNs) in MDA-MB231 cells. Materials and methodsAfter treating MCF-7 and MDA-MB231 cells with 18.9 μM and 13.4 μM of QC and QC-SLN for 48 h, respectively, cell viability, migration, sphere formation, protein expression of β-catenin, p-Smad 2 and 3, and gene expression of EMT and CSC markers were evaluated. ResultsThe QC-SLN with particle size of 154 nm, zeta potential of −27.7 mV, and encapsulation efficacy of 99.6% was found to be the most effective. Compared to QC, QC-SLN significantly reduced cell viability, migration, sphere formation, protein expression of β-catenin and p-Smad 2 and 3, and gene expression of CD44, zinc finger E-box binding homeobox 1 (ZEB1), vimentin, while increasing the gene expression of E-cadherin. ConclusionsOur findings demonstrate that SLNs improve the cytotoxic effect of QC in MDA-MB231 cells by increasing its bioavailability and inhibiting epithelial-mesenchymal transition (EMT), thereby effectively inhibiting CSC generation. Therefore, SLNs could be a promising new treatment for TNBC, but more in vivo studies are needed to confirm their efficacy.

Abstract 1234: Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way

Abstract The immune checkpoint molecule programmed cell death-ligand 1 (PD-L1, CD274) suppresses immunity when the cell surface PD-L1 is engaged by programmed death-1 (PD-1) on antitumor immune cells. Although PD-L1 has been established and targeted in cancer immunotherapy, the tumor-intrinsic role of PD-L1 is less appreciated in tumor biology and therapeutic development. In this study, we showed that PD-L1 facilitates epithelial-to-mesenchymal transition (EMT) in non-small cell lung cancer (NSCLC) cells by upregulating EMT transcription factors and mesenchymal markers, and downregulating epithelial marker. RNA-sequencing was performed on paired exogenously PD-L1-overexpressed A549 cells and wild-type cells, and PD-L1-depleted H460 cells and wild-type cells, and transcriptomic features associated with PD-L1 status were identified using Gene Set Enrichment Analysis (GSEA). In both the paired samples, PD-L1 expression was associated with EMT-related pathways. Subsequent in vitro experiment showed that exogenous overexpression of PD-L1 in A549 and H522 cells increased both mRNA and protein expression of Twist, Zeb1, Snail, Slug, Vimentin, Fibronectin, and N-Cadherin. Correspondingly, the transcription factors and mesenchymal markers were decreased in PD-L1-depleted H460 and H596 cells. Recombinant PD-1 treatment also increased the expression of EMT transcription factors and markers on A549 and H460 cells. Moreover, interferon-γ stimulation on tumor cells increased expression of EMT transcription factors and markers, which was suppressed by PD-L1-knockdown, suggesting that interferon-γ-induced PD-L1 contributes to EMT. Our findings suggest that PD-L1 facilitates NSCLC progression by promoting the EMT in interferon-γ-independent and dependent way, and this intrinsic PD-L1 function could be targeted for better clinical management of PD-L1-overexpressing NSCLCs. Citation Format: Hyein Jeong, Hyun Kyung Ahn, Sehui Kim, Yoon Kyung Jeon. Tumor cell-intrinsic PD-L1 promotes epithelial-to-mesenchymal transition in non-small cell lung cancer in interferon-g-independent and dependent way [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1234.

Abstract 428: The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression

Abstract Background: Tyrosine Kinase Inhibitors (TKIs) such as Erlotinib (ER) and Osimertinib (OR) are being used to treat lung cancer patients with EGFR mutations; however, patients develop resistance to these drugs within 10-18 months. EGFR-TKI resistance may be mediated by epithelial-mesenchymal transition (EMT), which causes a motile and invasive phenotype similar to mesenchymal cells. Sphingosine kinase 2 (SphK2), which is upregulated in Non-Small Cell Lung Cancer (NSCLC), phosphorylates sphingosine to sphingosine-1-phosphate (S1P) and increases cell proliferation, tumor survival and cell migration. The sphingolipid rheostat controls the cell fate by maintaining a balance between pro-survival and pro-apoptotic sphingolipids, which is regulated by SphK2. However, the role of SphK2 in TKI resistance and EMT is not well understood. Hypothesis: We hypothesize that the inhibition/downregulation of SphK2 will reverse EMT, reduce tumorigenicity and overcome ER and OR resistance. Study design and Results: We studied expression of SphK2 in both drug resistant (OR and ER) and drug sensitive (parental) H358, H2170, H3255 and PC9 NSCLC cell lines. In drug resistant cells, qPCR and immunoblotting studies showed that SphK2 was upregulated by 1.5-1.7 and 1.5-3.3 fold in ER and OR resistant cell lines respectively compared to parental cells. 48 hours after SphK2 knockdown with siRNA treatment, SphK2 was downregulated by 4.6-fold and EMT mediator E-cadherin expression was upregulated by 2.9-fold. EMT proteins Vimentin and N-cadherin were downregulated by 3.8 and 2.2 fold respectively (p≤0.01). Using an MTT assay, differences in percentage viability of cells with different treatments were calculated with respect to their viability in the presence of OR. We found that 81.43% of H358OR and 76.35% of the H3255OR cells were inhibited by a combination of Opaganib, a Sphk2 inhibitor, and OR, this is 27.74% and 12.28% higher compared to the additive effect of drug and inhibitor in H358OR and H3255 cells respectively. Furthermore, transwell migration assays, wound healing assays and spheroid assays were performed in OR resistant cells and the results indicated inhibition of the above assays by 94.97%, 82.36%, 88.67% respectively when treated with a combination of Opaganib and OR as compared to the diluent. This indicates the potential of SphK2 inhibition to overcome TKI resistance and EMT. To analyze the role of SphK2 expression in cancer progression, IHC was performed on 63 early and 48 late-stage lung cancer patient sections. We observed upregulation of SphK2 in late-stage lung cancer (p≤0.05), indicating its role in cancer progression. Conclusion: In this study, SphK2 was shown to decrease tumor survival, cancer cell proliferation, and expression of EMT markers. Thus, SphK2 may be a promising clinical target for overcoming EGFR-TKI resistance and NSCLC tumor progression. Citation Format: S M Nasifuzzaman, Namrata Dube, Katie Lam, Robert J. Hillwig, Neelu Puri. The role of sphingosine kinase 2 in overcoming EGFR-tyrosine kinase inhibitor resistance, EMT and NSCLC tumor progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 428.