GLP2-GLP2R signal affects the viability and EGFR-TKIs sensitivity of PC9 and HCC827 cells

Background The resistance to epidermal growth factor receptor (EGFR)- tyrosine kinase inhibitors (TKIs) therapy is currently the major clinical challenge in the treatment of lung cancer. This study aims to reveal the role of glucagon-like peptide (GLP) 2 and GLP-2 receptor (GLP2R) signaling on the EGFR-TKIs and cisplatin resistance of lung cancer cells. Methods The common differentially expressed genes in PC9 and HCC827 cells that were individually resistant to one of the three EGFR-TKIs (dacomitinib, osimertinib and afatinib) were screened. The data were from GSE168043 and GSE163913. The expression of GLP2R in drug-resistant cells was detected by western blot. The effect of GLP2R expression down- or up-regulation on resistance to dacomitinib, osimertinib, afatinib or cisplatin was measured by CCK-8 and flow cytometry assays. The long-acting analog of GLP-2, teduglutide, treated the parental cells. Results A total of 143 common differentially expressed genes were identified. Compared with the parent cells, the GLP2R expression in drug-resistant cell lines was significantly up-regulated. The exogenous expression of GLP2R in parental cells enhanced cell viability, while knockdown of GLP2R levels in drug-resistant cell lines inhibited cell viability. In addition, teduglutide treatment also enhanced the viability of lung cancer cells. Conclusion GLP2-GLP2R signal may change the sensitivity of cells to EGFR-TKIs and cisplatin. The development of GLP-2 or GLP2R inhibitors may be beneficial to the clinical treatment of lung cancer.

Impact of Cancer Stem Cells and Cancer Stem Cell-Driven Drug Resiliency in Lung Tumor: Options in Sight

Causing a high mortality rate worldwide, lung cancer remains an incurable malignancy resistant to conventional therapy. Despite the discovery of specific molecular targets and new treatment strategies, there remains a pressing need to develop more efficient therapy to further improve the management of this disease. Cancer stem cells (CSCs) are considered the root of sustained tumor growth. This consensus corroborates the CSC model asserting that a distinct subpopulation of malignant cells within a tumor drives and maintains tumor progression with high heterogeneity. Besides being highly tumorigenic, CSCs are highly refractory to standard drugs; therefore, cancer treatment should be focused on eliminating these cells. Herein, we present the current knowledge of the existence of CSCs, CSC-associated mechanisms of chemoresistance, the ability of CSCs to evade immune surveillance, and potential CSC inhibitors in lung cancer, to provide a wider insight to drive a more efficient elimination of this pro-oncogenic and treatment-resistant cell fraction.

LIM-Domain-Binding Protein 1 Mediates Cell Proliferation and Drug Resistance in Colorectal Cancer

Objective: It has been shown that LIM-domain-binding protein 1 (LDB1) is involved in the tumorigenesis of several cancers, but its function in colorectal cancer (CRC) has not been fully explained. This study is aimed to investigate whether LDB1 is involved in regulating the cell growth and drug sensitivity of CRC.Methods: To analyze the protein expression of LDB1 in CRC tissues, western blot was used. KM plotter and UALCAN databases were used to predict the prognosis of CRC patients with low or high LDB1 expression. To do the correlation analysis in CRC tissues, GEPIA database was used. CCK-8 assay and xenograft models were used to evaluate the effects of LDB1 in CRC cell growth. An oxaliplatin-resistant cell line was constructed to evaluate the effect of LDB1 in drug sensitivity of CRC cells.Results: Our current research confirmed that LDB1 was upregulated in CRC tumor tissues, and its elevation predicted a poor prognosis for CRC patients. LDB1 was also found positively correlated with CCNA1, BCL2 and BCLW, but negatively correlated with the pro-apoptotic signals (BID, BAX and BAK). Silence of LDB1 significantly inhibited CRC cell growth in vitro, and CRC cells with low expression of LDB1 had a lower tumorigenesis rate in tumor-bearing nude mice. Our experiments also showed that LDB1 silence enhanced the anti-tumor activity of oxaliplatin in CRC cells. The expression of LDB1 was also found increased in oxaliplatin-resistant CRC cell lines, and silence of LDB1 partly restored the antitumor effect of oxaliplatin in an oxaliplatin-resistant CRC cell line.Conclusion: Our current results revealed the roles of LDB1 in the growth and drug resistance of CRC cells, and may provide the new theoretical support for LDB1 as a potential target for the treatment of CRC in the future.

miR-489 Confines Uncontrolled Estrogen Signaling Through a Negative Feedback Mechanism and Regulates Tamoxifen Resistance in Breast Cancer

Background: Approximately 75% of diagnosed breast cancer tumors are estrogen-receptor (ER) positive tumors and are associated with better prognosis due to their response to hormonal therapies. However, around 40% of patients relapse after hormonal therapies. In the current study, we aim to evaluate miR-489 as a novel molecular target to combat tamoxifen resistance. Methods: Genomic analysis of gene expression profiles in primary breast cancers and tamoxifen resistant cell lines unveiled the potential role of miR-489 in regulation of estrogen signaling and development of tamoxifen resistance. We manipulated miR-489 expression in breast cancer cell lines by transient transfection of a miR-489 mimic or establishment of knockout cell lines using the CRISPR/Cas9 system to study the reciprocal regulation of miR-489 and estrogen/ER signaling pathways. Cell proliferation assays, Sphere-formation assays and flow cytometry analysis were conducted to investigate the role of miR-489 in estrogen-induced cell proliferation, cancer stem cell expansion and development of tamoxifen resistance. Results: miR-489 expression was significantly downregulated in tamoxifen-resistant cell lines. Low levels of miR-489 were associated with poor clinical outcomes in patients with hormone treatment. In vitro analysis showed that loss of miR-489 expression promoted tamoxifen resistance while overexpression of miR-489 in tamoxifen-resistant cells restored tamoxifen sensitivity. Mechanistically, we found that miR-489 is an estrogen regulated miRNA that negatively regulated estrogen receptor signaling by using at least the following two mechanisms: i) modulation of ER phosphorylation status by inhibiting MAPK and AKT kinase activities and downregulating SHP2 expression; ii) regulation of nucleus to cytosol translocation of estrogen receptor α (ERα) by decreasing p38 expression and consequently ER phosphorylation. In addition, miR-489 could break the positive feed-forward loop between the estrogen-ERα axis and p38 MAPK in breast cancer cells, which was necessary for its function as a transcription factor. Conclusion: Our study unveiled the underlying molecular mechanism by which miR-489 regulates estrogen signaling pathway through a negative feedback loop and uncovered its role in both the development of and overcoming of tamoxifen resistance in breast cancers.

Ad-VT enhances the sensitivity of chemotherapy-resistant lung adenocarcinoma cells to gemcitabine and paclitaxel in vitro and in vivo

SummaryBackground One of the main challenges in the clinical treatment of lung cancer is resistance to chemotherapeutic drugs. P-glycoprotein (P-gp)-mediated drug resistance is the main obstacle to successfully implementing microtubule-targeted tumor chemotherapy. Purpose In this study, we explored the effect of Ad-hTERTp-E1a-Apoptin (Ad-VT) on drug-resistant cell lines and the molecular mechanism by which Ad-VT combined with chemotherapy affects drug-resistant cells and parental cells. Methods In vitro, cell proliferation, colony formation, resistance index (RI), apoptosis and autophagy assays were performed. Protein expression was analyzed by Western blotting. Finally, a xenograft tumor model in nude mice was used to detect tumor growth and evaluate histological characteristics. Results Our results showed that Ad-VT had an obvious killing effect on A549, A549/GEM and A549/Paclitaxel cancer cells, and the sensitivity of drug-resistant cell lines to Ad-VT was significantly higher than that of parental A549 cells. Compared with A549 cells, A549/GEM and A549/Paclitaxel cells had higher autophagy levels and higher viral replication ability. Ad-VT decreased the levels of p-PI3k, p-Akt and p-mTOR and the expression of P-gp. In vivo, Ad-VT combined with chemotherapy can effectively inhibit the growth of chemotherapy-resistant tumors and prolong the survival of mice. Conclusions Thus, the combination of Ad-VT and chemotherapeutic drugs will be a promising strategy to overcome chemoresistance.

The Profile of MicroRNA Expression and Potential Role in the Regulation of Drug-Resistant Genes in Cisplatin- and Paclitaxel-Resistant Ovarian Cancer Cell Lines

Ovarian cancer is the most lethal gynecological malignancy. The high mortality results from late diagnosis and the development of drug resistance. Drug resistance results from changes in the expression of different drug-resistance genes that may be regulated miRNA. The main aim of our study was to detect changes in miRNA expression levels in two cisplatin (CIS) and two paclitaxel (PAC)—resistant variants of the A2780 drug-sensitive ovarian cancer cell line—by miRNA microarray. The next goal was to identify miRNAs responsible for the regulation of drug-resistance genes. We observed changes in the expression of 46 miRNA that may be related to drug resistance. The overexpression of miR-125b-5p, miR-99a-5p, miR-296-3p, and miR-887-3p and downregulation of miR-218-5p, miR-221-3p, and miR-222-3p was observed in both CIS-resistant cell lines. In both PAC-resistant cell lines, we observed the upregulation of miR-221-3p, miR-222-3p, and miR-4485, and decreased expression of miR-551b-3p, miR-551b-5p, and miR-218-5p. Analysis of targets suggest that expression of important drug-resistant genes like protein Tyrosine Phosphatase Receptor Type K (PTPRK), receptor tyrosine kinase—EPHA7, Semaphorin 3A (SEMA3A), or the ATP-binding cassette subfamily B member 1 gene (ABCB1) can be regulated by miRNA.

Comprehensive Molecular Landscape of Cetuximab Resistance in Head and Neck Cancer Cell Lines

Cetuximab is the sole anti-EGFR monoclonal antibody that is FDA approved to treat head and neck squamous cell carcinoma (HNSCC). However, no predictive biomarkers of cetuximab response are known for HNSCC. Herein, we address the molecular mechanisms underlying cetuximab resistance in an in vitro model. We established a cetuximab resistant model (FaDu), using increased cetuximab concentrations for more than eight months. The resistance and parental cells were evaluated for cell viability and functional assays. Protein expression was analyzed by Western blot and human cell surface panel by lyoplate. The mutational profile and copy number alterations (CNA) were analyzed using whole-exome sequencing (WES) and the NanoString platform. FaDu resistant clones exhibited at least two-fold higher IC50 compared to the parental cell line. WES showed relevant mutations in several cancer-related genes, and the comparative mRNA expression analysis showed 36 differentially expressed genes associated with EGFR tyrosine kinase inhibitors resistance, RAS, MAPK, and mTOR signaling. Importantly, we observed that overexpression of KRAS, RhoA, and CD44 was associated with cetuximab resistance. Protein analysis revealed EGFR phosphorylation inhibition and mTOR increase in resistant cells. Moreover, the resistant cell line demonstrated an aggressive phenotype with a significant increase in adhesion, the number of colonies, and migration rates. Overall, we identified several molecular alterations in the cetuximab resistant cell line that may constitute novel biomarkers of cetuximab response such as mTOR and RhoA overexpression. These findings indicate new strategies to overcome anti-EGFR resistance in HNSCC.

SORT1/LAMP2-mediated Extracellular Vesicle Secretion and Cell Adhesion Are Linked to Lenalidomide Resistance in Multiple Myeloma

Multiple myeloma (MM) is a hematopoietic malignancy whose prognosis has improved with the development of new agents such as lenalidomide over the last decade. However, long-term exposure to drugs induces the acquisition of resistance by MM cells and leads to treatment failure and poor prognosis. Here, we show the molecular and cellular mechanisms of lenalidomide resistance in MM. In a comparison between lenalidomide-resistant cell lines and the parental cell lines, the EV (Extracellular versicles) secretion and adherence abilities were significantly elevated in the resistant cells. Whole-transcriptome analysis revealed that the SORT1 and LAMP2 genes were key regulators of EV secretion. Silencing of these genes caused decreased EV secretion and loss of cell adhesion in the resistant cells, resulting in increased sensitivity to lenalidomide. Analysis of publicly available transcriptome data confirmed the relationship between genes related to EV secretion and cell adhesion and patient prognosis. Together, our findings reveal a novel mechanism of lenalidomide resistance in MM mediated by EV secretion and cell adhesion via SORT1 and LAMP2.

Nanophytotherapeutics for Cancer

Phytochemicals have been attributed beneficial health properties, mainly their anticancer potential. Cancer treatment seeks to shrink the tumor and kill cancer cells; however, the conventional treatment available frequently fails due to the emergence of drug-resistant cell lines. Plant-derived compounds have been studied for their potential anticancer effects or as adjuvant drug to conventional treatment. However, some of the physicochemical properties and stability characteristics of the phytocompounds generate biopharmaceuticals difficulties that limit their efficacy and clinical applications in oncology. In this sense, nanomedicine offers an alternative for the development of biocompatible, biodegradable, safe, and efficacy phytoformulations. Nanostructured delivery systems show immense potential in the bioavailability of phytodrugs by providing better alternatives to conventional dosage forms, through improving physicochemical and biopharmaceutical properties of the phytocompounds and along with it to enhance the therapeutic efficacy.

Biochemical and Cellular Characterization of New Radio-Resistant Cell Lines Reveals a Role of Natural Flavonoids to Bypass Senescence

Cancer is one of the main causes of death worldwide, and, among the most frequent cancer types, osteosarcoma accounts for 56% of bone neoplasms observed in children and colorectal cancer for 10.2% of tumors diagnosed in the adult population. A common and frequent hurdle in cancer treatment is the emergence of resistance to chemo- and radiotherapy whose biological causes are largely unknown. In the present work, human osteosarcoma (SAOS) and colorectal adenocarcinoma (HT29) cell lines were γ-irradiated at doses mimicking the sub-lethal irradiation in clinical settings to obtain two radio-resistant cellular sub-populations named SAOS400 and HT500, respectively. Since “therapy-induced senescence” (TIS) is often associated with tumor response to radiotherapy in cancer cells, we measured specific cellular and biochemical markers of senescence in SAOS400 and HT500 cells. In detail, both cell lines were characterized by a higher level of expression of cyclin-dependent kinase inhibitors p16INK4 and p21CIP1 and increased positivity to SAβ-gal (senescence-associated β-galactosidase) with respect to parental cells. Moreover, the intracellular levels of reactive oxygen species in the resistant cells were significantly lower compared to the parental counterparts. Subsequently, we demonstrated that senolytic agents were able to sensitize SAOS400 and HT500 to cell death induced by γ-irradiation. Employing two natural flavonoids, fisetin and quercetin, and a BH3-mimetic, ABT-263/navitoclax, we observed that their association with γ-irradiation significantly reduced the expression of p16INK4, p21CIP1 and synergistically (combination index < 1) increased cell death compared to radiation mono-alone treatments. The present results reinforce the potential role of senolytics as adjuvant agents in cancer therapy.