Constitutive activation of the epidermal growth factor receptor (EGFR) because of somatic mutations of the EGFR gene is commonly observed in tumors of non–small cell lung cancer (NSCLC) patients. Consequently, tyrosine kinase inhibitors (TKI) targeting the EGFR are among the most effective therapies for patients with sensitizing EGFR mutations. Clinical responses to the EGFR-targeting TKIs are evaluated through 2-[¹⁸F]fluoro-2-deoxy-glucose (¹⁸FDG)-PET uptake, which is decreased in patients responding favorably to therapy and is positively correlated with survival. Recent studies have reported that EGFR signaling drives glucose metabolism in NSCLC cells; however, the precise downstream effectors required for this EGFR-driven metabolic effect are largely unknown. 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) is an essential glycolytic regulator that is consistently overexpressed in lung cancer. Here, we found that PFKFB3 is an essential target of EGFR signaling and that PFKFB3 activation is required for glycolysis stimulation upon EGFR activation. We demonstrate that exposing NSCLC cells harboring either WT or mutated EGFR to EGF rapidly increases PFKFB3 phosphorylation, expression, and activity and that PFKFB3 inhibition markedly reduces the EGF-mediated increase in glycolysis. Furthermore, we found that prolonged NSCLC cell exposure to the TKI erlotinib drives PFKFB3 expression and that chemical PFKFB3 inhibition synergizes with erlotinib in increasing erlotinib's anti-proliferative activity in NSCLC cells. We conclude that PFKFB3 has a key role in mediating glucose metabolism and survival of NSCLC cells in response to EGFR signaling. These results support the potential clinical utility of using PFKFB3 inhibitors in combination with EGFR-TKIs to manage NSCLC.

Inhibition of ERN1/IRE1α (endoplasmic reticulum to nucleus signaling 1/inositol requiring enzyme-1α), the major signaling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of transcription factors such as E2F8 (E2F transcription factor 8), EPAS1 (endothelial PAS domain protein 1), TBX3 (T-box 3), ATF3 (activating transcription factor 3), FOXF1 (forkhead box F1), and HOXC6 (homeobox C6) in U87 glioma cells overexpressing dominant-negative ERN1/IRE1α defective in endoribonuclease (dnr-ERN1) as well as defective in both kinase and endonuclease (dn-ERN1) activity of ERN1/IRE1α. We have demonstrated that the expression of all studied genes is decreased at the mRNA level in cells with modified ERN1/IRE1α; TBX3, however, is increased in these cells as compared to control glioma cells. Changes in protein levels of E2F8, HOXC6, ATF3, and TBX3 corresponded to changes in mRNAs levels. We also found that two mutated ERN1/IRE1α have differential effects on the expression of studied transcripts. The presence of kinase and endonuclease deficient ERN1/IRE1α in glioma cells had a less profound effect on the expression of E2F8, HOXC6, and TBX3 genes than the blockade of the endoribonuclease activity of ERN1/IRE1α alone. Kinase and endonuclease deficient ERN1/IRE1α suppresses ATF3 and FOXF1 gene expressions, while inhibition of only endoribonuclease of ERN1/IRE1α leads to the up-regulation of these gene transcripts. The present study demonstrates that fine-tuning of the expression of proliferation related genes is regulated by ERN1/IRE1α an effector of endoplasmic reticulum stress. Inhibition of ERN1/IRE1α, especially its endoribonuclease activity, correlates with deregulation of proliferation related genes and thus slower tumor growth.

While clinical responses to palbociclib have been promising, metastatic breast cancer remains incurable due to the development of resistance. We generated estrogen receptor-positive (ER+) and ER-negative (ER−) cell line models and determined their permissiveness and cellular responses to an oncolytic adenovirus (OAd) known as Ad5/3-delta24. Analysis of ER+ and ER− palbociclib-resistant cells revealed two clearly distinguishable responses to the OAd. While ER+ palbociclib-resistant cells displayed a hypersensitive phenotype to the effects of the OAd, ER− palbociclib-resistant cells showed a resistant phenotype to the OAd. Hypersensitivity to the OAd in ER+ palbociclib-resistant cells correlated with a decrease in type I interferon (IFN) signaling, an increase in viral entry receptor expression, and an increase in cyclin E expression. OAd resistance in ER− palbociclib-resistant cells correlated with an increase in type I IFN signaling and a marked decrease in viral entry receptor. Using the OAd as monotherapy caused significant cytotoxicity to both ER+ and ER− palbociclib-sensitive cell lines. However, the addition of palbociclib increased the oncolytic activity of the OAd only in ER+ palbociclib-sensitive cells. Our studies provide a mechanistic base for a novel anti-cancer regimen composed of an OAd in combination with palbociclib for the treatment of ER+ breast cancer.

Acquired resistance to cyclin-dependent kinases 4 and 6 (CDK4/6) inhibition in estrogen receptor-positive (ER+) breast cancer remains a significant clinical challenge. Efforts to uncover the mechanisms underlying resistance are needed to establish clinically actionable targets effective against resistant tumors. In this study, we sought to identify differentially expressed genes (DEGs) associated with acquired resistance to palbociclib in ER+ breast cancer. We performed next-generation transcriptomic RNA sequencing (RNA-seq) and pathway analysis in ER+ MCF7 palbociclib-sensitive (MCF7/pS) and MCF7 palbociclib-resistant (MCF7/pR) cells. We identified 2183 up-regulated and 1548 down-regulated transcripts in MCF7/pR compared to MCF7/pS cells. Functional analysis of the DEGs using Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database identified several pathways associated with breast cancer, including ‘cell cycle’, ‘DNA replication’, ‘DNA repair’ and ‘autophagy’. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with deregulation of several key canonical and metabolic pathways. Further studies are needed to determine the utility of these DEGs and pathways as therapeutics targets against ER+ palbociclib-resistant breast cancer.

Inhibition of ERN1 (endoplasmic reticulum to nuclei 1), the major signalling pathway of endoplasmic reticulum stress, significantly decreases tumor growth. We have studied the expression of tumor protein 53 (TP53)- related genes such as TOPORS (topoisomerase I binding, arginine/serine-rich, E3 ubiquitin protein ligase), TP53BP1 (TP53 binding protein 1), TP53BP2, SESN1 (sestrin 1), NME6 (non-metastatic cells 6), and ZMAT3 (zinc finger, Matrin-type 3) in glioma cells expressing dominantnegative ERN1 under baseline and hypoxic conditions. We demonstrated that inhibition of ERN1 function in U87 glioma cells resulted in increased expression of RYBP, TP53BP2, and SESN1 genes, but decreased expression of TP53BP1, TOPORS, NME6, and ZMAT3 genes. Moreover, inhibition of ERN1 affected hypoxia-mediated changes in expression of TP53-related genes and their magnitude. Indeed, hypoxia has no effect on expression of TP53BP1 and SESN1 in control cells, while resulted in increased expression of these genes in cells with inhibited ERN1 function. Magnitude of hypoxia-mediated changes in expression levels of RYBP and TP53BP2 was gene specific and more robust in the case of TP53BP2. Hypoxiamediated decrease in expression levels of TOPORS was more prominent if ERN1 was inhibited. Present study demonstrates that fine-tuning of the expression of TP53- associated genes depends upon endoplasmic reticulum stress signaling under normal and hypoxic conditions. Inhibition of ERN1 branch of endoplasmic reticulum stress response correlates with deregulation of p53 signaling and slower tumor growth.

Purpose: The management of triple-negative breast cancer (TNBC) remains a significant clinical challenge due to the lack of effective targeted therapies. Inhibitors of the cyclin-dependent kinases 4 and 6 (CDK4/6) are emerging as promising therapeutic agents against TNBC; however, cells can rapidly acquire resistance through multiple mechanisms that are yet to be identified. Therefore, determining the mechanisms underlying resistance to CDK4/6 inhibition is crucial to develop combination therapies that can extend the efficacy of the CDK4/6 inhibitors or delay resistance. This study aims to identify differentially expressed genes (DEG) associated with acquired resistance to palbociclib in ER− breast cancer cells. Methods: We performed next-generation transcriptomic sequencing (RNA-seq) and pathway analysis in ER− MDA-MB-231 palbociclib-sensitive (231/pS) and palbociclib-resistant (231/pR) cells. Results: We identified 2247 up-regulated and 1427 down-regulated transcripts in 231/pR compared to 231/pS cells. DEGs were subjected to functional analysis using Gene Ontology (GO) and the KEGG database which identified many transduction pathways associated with breast cancer, including the PI3K/AKT, PTEN and mTOR pathways. Additionally, Ingenuity Pathway Analysis (IPA) revealed that resistance to palbociclib is closely associated with altered cholesterol and fatty acid biosynthesis suggesting that resistance to palbociclib may be dependent on lipid metabolic reprograming. Conclusion: This study provides evidence that lipid metabolism is altered in TNBC with acquired resistance to palbociclib. Further studies are needed to determine if the observed lipid metabolic rewiring can be exploited to overcome therapy resistance in TNBC.