From Microbes to Medicine: Bacteriotherapy's Emerging Role in Cancer Treatment Strategies

Background/Aims: Our group reported that cinnamaldehyde derivative, (E)-4-((2-(3-oxopop-1-enyl)phenoxy)methyl)pyridinium malonic acid (CB-PIC) induced apoptosis in hypoxic SW620 colorectal cancer cells via activation of AMP-activated protein kinase (AMPK) and extracellular signal regulated kinase (ERK). Herein, sensitizing effect of CB-PIC was investigated in resistant cancer cells such as paclitaxel (PT) resistant lung cancer cells (H460/PT), and Adriamycin (Adr) resistant breast cancer (MCF7/Adr) and colon cancer (HCT15/cos) cells. Methods: Various drug resistant cell lines were treated with CB-PIC, and the signalling pathway and functional assay were explored by Western blot, Rhodamine assay, FACS, RT-PCR and MTT assay. Results: We found that CB-PIC effectively exerted cytotoxicity, increased sub G1 population and the cleaved form of poly (ADP-ribose) polymerase (PARP) and caspase 9 in drug resistant cancer cells. Furthermore, CB-PIC sensitized resistant cancer cells to adriamycin via downregulation of survival proteins such as survivin, Bcl-xL and Bcl-2, along with MDR1 suppression leading to accumulation of drug in the intracellular region. Of note, CB-PIC transcriptionally decreased MDR1 expression via suppression of STAT3 and AKT signalling in three resistant cancer cells with highly expressed P-glycoprotein. Nonetheless, CB-PIC did not affect transport activity of P-glycoprotein in a short time efflux assay, while epigallocatechin gallate (EGCG) accumulated Rhodamine 123 into intracellular region of cell by direct inhibition of MDR1 transport activity. Conclusions: These data demonstrate that CB-PIC suppresses the P-glycoprotein expression through inhibition of STAT3 and AKT signalling to overcome drug resistance in chemo-resistant cancer cells as a potent chemotherapeutic sensitizer.

The process of tumor metastasis is highly complex and involves dissemination of tumor cells from the primary tumor through the vascular system. However, most of the cancer cells, particularly epithelial cancer cells, have very low survival rates in circulation and undergo rapid anoikis. Tumor cells that acquire malignant potential have developed mechanisms to resist anoikis and thereby survive while travelling though the lymphatic or circulatory systems. Recently, we have shown that IL-6 promotes epithelial-mesenchymal transition (EMT) and tumor metastasis. A number of studies have shown that FAK signaling pathways play an important role in inducing anoikis resistance in tumor cells. In this study, we examined if IL-6 promoted tumor metastasis by enhancing stemness and anoikis resistance in cancer cells via the activation of FAK signaling. Our results demonstrate that IL-6 significantly enhanced stemness phenotype in cancer cells as measured by tumorsphere formation and ALDH positive cells by upregulating nanog protein levels. Nanog knockdown significantly reversed IL-6-mediated cancer stem cell (CSC) phenotype. IL-6 treatment of cancer cells induced FAK phosphorylation in a time dependent manner. Interestingly, FAK knockdown did not significantly decrease nanog mRNA levels but markedly decreased nanog protein levels. FAK depletion completely blocked IL-6-mediated nanog phosphorylation and enhanced nanog protein ubiquitination and degradation. These results suggest that FAK may be modulating nanog protein levels by stabilization nanog protein through phosphorylation. Furthermore, FAK knockdown significantly decreased IL-6-mediated tumorsphere formation, anoikis resistance and tumor metastasis. Taken together, our results show that IL-6 promotes tumor metastasis by enhancing stem cell phenotype and anoikis resistance in head and neck cancer cells via FAK activation and nanog protein stabilization. Citation Format: Arti Yadav, Bhavna Kumar, Pawan Kumar. IL-6 promotes tumor metastasis by promoting anoikis resistance and stemness phenotype in cancer cells via the activation of FAK and nanog stabilization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3447.

Simple SummaryAcquired resistance to chemotherapy by cancer cells is the predominant factor in chemotherapy failure, which ultimately leads to disease progression and death. Recent studies have presented compelling evidence of the various mechanisms and pathways through which cancer cells have developed resistance to drugs. This review summarises the mechanisms pertaining to 5-FU resistance and discusses ongoing efforts to prevent chemotherapy resistance in cancer cells and to re-sensitise them to cancer drugs.5-Fluorouracil (5-FU) plus leucovorin (LV) remain as the mainstay standard adjuvant chemotherapy treatment for early stage colon cancer, and the preferred first-line option for metastatic colon cancer patients in combination with oxaliplatin in FOLFOX, or irinotecan in FOLFIRI regimens. Despite treatment success to a certain extent, the incidence of chemotherapy failure attributed to chemotherapy resistance is still reported in many patients. This resistance, which can be defined by tumor tolerance against chemotherapy, either intrinsic or acquired, is primarily driven by the dysregulation of various components in distinct pathways. In recent years, it has been established that the incidence of 5-FU resistance, akin to multidrug resistance, can be attributed to the alterations in drug transport, evasion of apoptosis, changes in the cell cycle and DNA-damage repair machinery, regulation of autophagy, epithelial-to-mesenchymal transition, cancer stem cell involvement, tumor microenvironment interactions, miRNA dysregulations, epigenetic alterations, as well as redox imbalances. Certain resistance mechanisms that are 5-FU-specific have also been ascertained to include the upregulation of thymidylate synthase, dihydropyrimidine dehydrogenase, methylenetetrahydrofolate reductase, and the downregulation of thymidine phosphorylase. Indeed, the successful modulation of these mechanisms have been the game plan of numerous studies that had employed small molecule inhibitors, plant-based small molecules, and non-coding RNA regulators to effectively reverse 5-FU resistance in colon cancer cells. It is hoped that these studies would provide fundamental knowledge to further our understanding prior developing novel drugs in the near future that would synergistically work with 5-FU to potentiate its antitumor effects and improve the patient’s overall survival.

We investigated possible conditions or drugs that might enhance the sensitivity of anti-mitotic drug-resistant cancer cells. We particularly focused on identifying mechanisms or drugs that could sensitize P-glycoprotein (P-gp)-overexpressing resistant KBV20C cancer cells. Our approach utilized repositioning drugs, which are already used in clinics, because once their sensitization mechanisms on resistant cancer cells are known, they would be readily applied without further toxicity studies. Selenium-derived drugs such as selenate, selenite, selenomethionine (SeMet), methyl-selenocysteine (MSC), and methaneselenic acid (MSA) have been shown to have anti-cancer properties clinically. The type of selenium-derived drug that can specifically sensitize P-gp-overexpressing resistant KBV20C cancer cells was investigated for further application in the clinical settings. We recently reported five selenium-derived drugs that could sensitize both resistant KBV20C and KB parent sensitive cancer cells without P-gp inhibition. Among these five drugs, our study highlights the unprecedented finding of the selective sensitization ability of selenate against P-gp-overexpressed resistant KBV20C cells. Detailed analysis indicates that selenate is a resistant cancer cell-specific sensitizing drug that increases apoptosis via G2-phase cell cycle arrest. These results may help improve chemotherapeutic treatments based on selenium-derived drugs for cancer patients who develop resistance to anti-mitotic drugs.

Simple SummaryUsing a cell culture model of resistant breast cancer cells with the phenotype that is often responsible for the early relapse of triple-negative breast cancer, namely, the persistence of these cells in reversible quiescence under a variety of challenges, we found that reprogramming the epigenome by treatment with JIB-04, a small-molecule inhibitor of Jumonji-family histone demethylases, sensitized resistant cells. We used this model of deep intrinsic resistance featuring many molecular mechanisms of achieving this phenotype to perform lengthy evaluations of less cytotoxic doses of JIB-04. We found that resistant cells derived from triple-negative inflammatory breast cancer cell lines were either much more sensitive to JIB-04 than the parental cell line or altered by the treatment such that they became sensitive to the chemotherapeutic drugs paclitaxel and doxorubicin. Notably, JIB-04 exposure increased PD-L1 expression in cancer cells, which means that JIB-04 may have clinical applications in improving the responses of triple-negative breast cancer to anti-PD-L1 therapy.In the present study, we evaluated JIB-04, a small-molecule epigenetic inhibitor initially discovered to inhibit cancer growth, to determine its ability to affect deep intrinsic resistance in a breast cancer model. The model was based on a function-based approach to the selection of cancer cells in a cell culture that can survive a variety of challenges in prolonged, but reversible, quiescence. These resistant cancer cells possessed a variety of mechanisms, including modifications of the epigenome and transcriptome, for generating a high degree of cellular heterogeneity. We found that long pretreatment with JIB-04 sensitized resistant triple-negative inflammatory breast cancer cells and their parental cell line SUM149 to the chemotherapeutic drugs doxorubicin and paclitaxel. Resistant cancer cells derived from another inflammatory breast cancer cell line, FC-IBC02, were considerably more sensitive to JIB-04 than the parental cell line. Investigating a mechanism of sensitization, we found that JIB-04 exposure increased the expression of PD-L1 in resistant cells, suggesting that JIB-04 may also sensitize resistant breast cancer cells to anti-PD-L1 immune therapy. Finally, these results support the usefulness of a cell culture-based experimental strategy for evaluating anticancer agents, such as JIB-04, that may halt cancer evolution and prevent the development of cancer resistance to currently used therapies.

5-Oxo-hexahydroquinoline derivatives as modulators of P-gp, MRP1 and BCRP transporters to overcome multidrug resistance in cancer cells

Desmoplasia of Pancreatic Ductal Adenocarcinoma

BackgroundTumor heterogeneity is one of the key factors leading to chemo-resistance relapse. It remains unknown how resistant cancer cells influence sensitive cells during cohabitation and growth within a heterogenous tumors. The goal of our study was to identify driving factors that mediate the interactions between resistant and sensitive cancer cells and to determine the effects of cohabitation on both phenotypes.MethodsWe used isogenic ovarian cancer (OC) cell lines pairs, sensitive and resistant to platinum: OVCAR5 vs. OVCAR5 CisR and PE01 vs. PE04, respectively, to perform long term direct culture and to study the phenotypical changes of the interaction of these cells.ResultsLong term direct co-culture of sensitive and resistant OC cells promoted proliferation (p < 0.001) of sensitive cells and increased the proportion of cells in the G1 and S cell cycle phase in both PE01 and OVCAR5 cells. Direct co-culture led to a decrease in the IC50 to platinum in the cisplatin-sensitive cells (5.92 µM to 2.79 µM for PE01, and from 2.05 µM to 1.51 µM for OVCAR5). RNAseq analysis of co-cultured cells showed enrichment of Cell Cycle Control, Cyclins and Cell Cycle Regulation pathways. The transcription factor E2F1 was predicted as the main effector responsible for the transcriptomic changes in sensitive cells. Western blot and qRT-PCR confirmed upregulation of E2F1 in co-cultured vs monoculture. Furthermore, an E2F1 inhibitor reverted the increase in proliferation rate induced by co-culture to baseline levels.ConclusionOur data suggest that long term cohabitation of chemo-sensitive and -resistant cancer cells drive sensitive cells to a higher proliferative state, more responsive to platinum. Our results reveal an unexpected effect caused by direct interactions between cancer cells with different proliferative rates and levels of platinum resistance, modelling competition between cells in heterogeneous tumors.

P-glycoprotein (Pgp) overexpression is frequently associated with multidrug resistance in cancer cells. Pgp is an efflux pump and is encoded by human mdr1 gene. The expression of mdr1 may be regulated by promoter methylation catalyzed by DNA methyltransferases (DNMTs). S-adenosyl-L-methionine (SAMe) is an important cofactor of DNMTs. Although SAMe has been shown to regulate promoter methylation of various genes, the effect of SAMe on Pgp expression and chemosensitivity of cancer cells are however not known. The aim of the present study is to examine the role of SAMe in the regulation of Pgp expression and drug sensitivity of human cancer cells. By qRT-PCR and Western blot analyses, incubation with 1mg/ml SAMe for 24h was found to increase mdr1 mRNA and Pgp protein levels in human hepatocellular carcinoma HepG2 cells, human colorectal adenocarcinoma CaCO-2 cells and human leukemia Jurkat-T cells. Methylation specific PCR and bisulfite DNA sequencing analyses revealed that the SAMe treatment induces hypomethylation of mdr1 promoter. Moreover, SAMe decreased intracellular doxorubicin accumulation and increased doxorubicin resistance in human cancer cells as assessed by doxorubicin accumulation assay and MTT assay. Furthermore, in human colorectal carcinoma HCT-116 parent cells and DNMTs knock-out sublines, SAMe induced Pgp expression in HCT-116 parent and DNMT3b knock-out subline but not in DNMT1 and DNMT1/DNMT3b double knockout sublines. The results from the present study therefore suggest that SAMe induces hypomethylation of mdr1 promoter, Pgp expression, and thus drug resistance in human cancer cells. Furthermore, DNMT1 appeared to be important in the effect of SAMe on mdr1. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1702. doi:10.1158/1538-7445.AM2011-1702

Apoptosis resistance in cancer cells adapted to acidic microenvironments poses a challenge for effective treatment. This study investigated the potential use of caffeic acid as an adjunct therapy to overcome drug resistance in colorectal cancer cells under acidic conditions. Long-term exposure to low-pH conditions induced resistance in HCT116 colorectal cancer cells. The effects of caffeic acid on proliferation, clonogenicity, and apoptosis induction were assessed alone and in combination with oxaliplatin and 5-Fluorouracil. The signaling pathways involved in drug resistance were examined by assessing the activities of PI3K/Akt and ERK1/2. Caffeic acid inhibited the proliferation and clonogenicity of acid-adapted cancer cells, and enhanced apoptosis when combined with anticancer drugs. Mechanistically, caffeic acid attenuated the hyperactivation of the PI3K/Akt and ERK1/2 signaling pathways associated with drug resistance. Caffeic acid is a promising therapeutic agent for targeting resistant cancer cells in acidic microenvironments. Its ability to inhibit proliferation, sensitize cells to apoptosis, and modulate signaling pathways highlights its potential for overcoming drug resistance in cancer therapy.

Simple SummaryCell culture models of cancer typically favor proliferative but therapy-sensitive cells because body-like selection pressures are absent. To address this limitation, we previously described a function-based selection strategy to model deep intrinsic resistance in cultures of triple-negative breast cancer cells. Our methods were designed to reveal therapy-resistant, adaptable cancer cells that opportunistically switch between quiescence and proliferation. To determine the validity of this approach in identifying noncytotoxic drugs that could inhibit highly resistant breast cancer cells, we used our novel cell culture model to evaluate a well-known BET bromodomain inhibitor, JQ1, which modulates the cancer epigenome. JQ1 has been found to inhibit resistant cancer cells in several cancer types, including breast cancer. Low-dose JQ1 inhibited the growth of highly adaptable/resistant breast cancer cells in our cell culture model. Our results support the validity of a cell culture-based approach for modeling cancer.We treated highly metabolically adaptable (SUM149-MA) triple-negative inflammatory breast cancer cells and their control parental SUM149-Luc cell line with JQ1 for long periods to determine its efficacy at inhibiting therapy-resistant cells. After 20 days of treatment with 1–2 µM of JQ1, which killed majority of cells in the parental cell line, a large number of SUM149-MA cells survived, consistent with their pan-resistant nature. Interestingly, though, the JQ1 treatment sensitized resistant cancer cells in both the SUM149-MA and SUM149-Luc cell lines to subsequent treatment with doxorubicin and paclitaxel. To measure JQ1-mediated sensitization of resistant cancer cells, we first eradicated approximately 99% of relatively chemotherapy-sensitive cancer cells in culture dishes by long treatments with doxorubicin or paclitaxel, and then analyzed the remaining resistant cells for survival and growth into colonies. In addition, combination, rather than sequential, treatment with JQ1 and doxorubicin was also effective in overcoming resistance. Notably, Western blotting showed that JQ1-treated cancer cells had significantly lower levels of PD-L1 protein than did untreated cells, indicating that JQ1 treatment may reduce tumor-mediated immune suppression and improve the response to immunotherapy targeting PD-L1. Finally, JQ1 treatment with a low 62.5 nM dose sensitized another resistant cell line, FC-IBC02-MA, to treatment with doxorubicin and paclitaxel.

Lung Cancer ManagementVol. 6, No. 4 EditorialFree AccessNRG1: a cinderella fusion in lung cancer?Lucia Anna Muscarella & Antonio RossiLucia Anna Muscarella Laboratory of Oncology, Scientific Institute for Research & Health Care (IRCCS) 'Casa Sollievo della Sofferenza', San Giovanni Rotondo (FG), Italy & Antonio Rossi*Author for correspondence: Tel.: +39 0882 410 716; Fax: +39 0882 204 095; E-mail Address: arossi_it@yahoo.it Division of Medical Oncology, Scientific Institute for Research & Health Care (IRCCS), Casa Sollievo della Sofferenza Hospital, Viale Cappuccini 1, 71013, San Giovanni Rotondo (FG), ItalyPublished Online:5 Jan 2018https://doi.org/10.2217/lmt-2017-0018AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: ErbBNRG1 fusionNSCLCThe amazing bridge between gene fusions and lung cancer has been greatly consolidated during the last decades. The identification of ALK and ROS1 rearrangements gives the great opportunity to rewrite the standard-of-care for a portion of advanced non-small-cell lung cancer (NSCLC) patients. Despite their very low incidence, these well-known genomic rearrangements share common features strictly associated to specific phenotypes of lung adenocarcinoma. This makes them ideal for diagnostic procedures, patients' stratification and therapies [1,2]. In this scenario, the finding of further intriguing targetable gene fusions, despite with a low incidence (1–2%) in NSCLC, is now increasing and it is watched with attention by the clinical community [3].Recently, the NRG1 gene has been described as a new molecular feature of NSCLC [4]. The NRG1 gene is located at the long arm of chromosome 10 (10q23.1 region) and encodes for the neuregulin 1, a growth factor belonging to the complex family of proteins also called heregulins. These proteins are structurally related to the stimulation of ERBB receptors tyrosine kinase activity and EGF signals. Specifically, the NRG1-receptor binding induces the phosphorylation of the intrinsic kinase domains of ERBB3 and stimulates its dimerization with ERBB2 receptor and the activation of the downstream PI3K-AKT and MAPK pathways [5].The neuronal isoform NRG1 III-β3 is generally not expressed in normal lung tissue, but it was found to be ectopically activated in lung tumor cells by NRG1 genomic rearrangements involving mainly CD74 and SLC3A2 genes [6]. It is the first fusion associated with the mucinous subtype of lung adenocarcinoma with an occurrence ranging from 8 to 27%. By contrast, NRG1-gene fusions occur in 1–2% of NSCLC and occasionally reported in other solid tumors [7]. It has been more extensively investigated in Asian than Caucasian lung cancer patients in whom it has been not yet fully elucidated and remains only partially understood [6].ERBB signaling is one of the most deregulated lung cancer cascades. ERBB3 is not frequently affected by mutations or amplifications in lung cancer. Furthermore, MET or HER2 amplifications, which represent additional mechanisms of ERBB3 activation in lung tumors, rarely occur. Thus, the overproduction of NRG1 ligands could represent one of the leading mechanisms by which lung cancer cells aberrantly activate ERBB3-related receptor tyrosine kinase signaling.The first suggestion of a real clinical utility of NRG1 fusion comes by analyzing a large sample of mucinous lung adenocarcinoma in Asians for the NRG1 breaks. In fact, patients with stage I disease harboring tumors with NRG1 fusions showed an inferior overall survival and a trend toward a shorter disease-free survival compared with those without NRG1 fusions [8]. The powerfulness of this fusion has been highlighted in vitro. In fact, the expression of CD74–NRG1 fusion gene is able to promote cancer stem cell properties and it is involved in stem cell function of several types of cancers, including lung cancer. These data imply the existence of a mechanism by which the activated ERBB receptors contribute to the acquisition of cancer stem cell-like characteristics together with the ability of cancer cells to develop a resistance to chemotherapy [9]. Moreover, in the absence of RAS pathway mutations, NRG1 overexpression can play a major role in the primary cetuximab resistance in colon cancer cells and in primary resistance to trastuzumab in HER2 overexpressing breast cancer cells [10,11].Of interest, NRG1 fusions have been proved to be coexistent with ALK fusion or RAS mutation in NSCLC patients, both in primary and in metastatic sites of lung tumors [12–14]. Moreover, it has been recently considered as a potential mechanism of resistance after treatment with tyrosine kinase inhibitors in ALK-rearranged NSCLC cell lines. In fact, by using primary cultures of cancer cells from pleural effusion of an ALK-positive lung cancer patient, the increase of the NRG1 ligand levels and the consequent activation of ERBB3 pathway has been directly related to resistance to crizotinib treatment [15]. This assumption has been further confirmed showing that, under treatment with second-generation ALK inhibitors, NSCLC cells activated the EGFR family pathways directly through the NRG1–ERBB3–EGFR activation axis [16]. In support to these in vitro reports, the onset of the SLC3A2–NRG1 gene fusion during the natural history of two invasive mucinous lung adenocarcinoma in Asiatic patients has been described. These heavily pretreated patients received the combination of lumretuzumab, a monoclonal anti-ERBB3 antibody, plus erlotinib, an anti-EGFR small molecule, showing tumor shrinkage [17]. Furthermore, the use of afatinib, a pan-ERBB-family kinase inhibitor, in NRG1-positive samples resulted in a surprisingly durable response in patients with lung adenocarcinoma and cholangiocarcinoma [7,18].ERBB3 overexpression actually represents one of the targets of greater interest for the current pharmacological studies. In fact, its activation through phosphorylation has been detected in various cancers including metastatic lung carcinoma in presence of acquired resistance to other ERBB family inhibitors [19]. Moreover, due to its inactive tyrosine kinase domain, ERBB3 has been reported to have a critical role in the dimerization process in the context of acquired de novo resistance to ERBB3 targeted therapies. However, despite the well-tested efficacy of EGFR and HER2 inhibitors, ERBB3 specific upregulation has not been yet targeted with clear clinical efficacy. Of all the anti-ERBB3 agents, patritumab is in advanced clinical development, being currently investigated in a Phase III trial for treatment of NSCLC (JapicCTI-101262) [20]. Additional trials are investigating neratinib, alone and in combination with temsirolimus (NCT01827267) and trastuzumab emtansine (T-DM1; NCT02289833), in HER2 molecular profiled advanced NSCLC. Of great interest is MM-121. An ongoing open-label trial is investigating MM-121, a fully human monoclonal antibody targeting specifically ERBB3, in combination with docetaxel or pemetrexed compared with docetaxel or pemetrexed alone, in patients with heregulin-positive, advanced NSCLC with primary end point overall survival (NCT02387216).Overall, NRG1 fusions could represent new potential molecular alterations able to predict the therapy activity in a specific lung adenocarcinoma subtype. Since NRG1 fusions act through the activation of the ERBB receptor, blocking the activity of the NRG1–ERBB–PI3K–AKT pathway might be the best strategy for the treatment of NRG1-fused tumors.Financial & competing interests disclosureThis work was supported by the Italian Ministry of Health (Ricerca Corrente, RC1703LO41 to LA Muscarella and RC1703ON39 to A Rossi), by the '5x1000' voluntary contributions and by the AIRC/MFAG grant 12983 (to LA Muscarella) and FBNC Grant 2015–2016 to LA Muscarella. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.References1 Kwak El, Bang YJ, Camidge DR et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N. Engl. J. Med. 363(18), 1693–1703 (2010).Crossref, Medline, CAS, Google Scholar2 Davies KD, Le AT, Theodoro MF et al. Identifying and targeting ROS1 gene fusions in non-small-cell lung cancer. Clin. Cancer Res. 18(17), 4570–4579 (2012).Crossref, Medline, CAS, Google Scholar3 Schram AM, Chang MT, Jonhsson P, Drilon A. Fusions in solid tumours: diagnostic strategies, targeted therapy and acquired resistance. Nat. Rev. Clin. Oncol. doi: 10.1038/nrclinonc.2017.127 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar4 Fernandez-Cuesta L, Plenker D, Osada H et al. CD74–NRG1 fusions in lung adenocarcinoma. Cancer Discov. 4(4), 415–422 (2014).Crossref, Medline, CAS, Google Scholar5 Yarden Y, Pines G. The ERBB network: at last, cancer therapy meets systems biology. Nat. Rev. Cancer 12(8), 553–563 (2012).Crossref, Medline, CAS, Google Scholar6 Trombetta D, Rossi A, Fabrizio FP et al. NRG1–ErbB lost in translation: a new paradigm for lung cancer? Curr. Med. Chem. doi: 10.2174/0929867324666170911170554 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar7 Jones MR, Lim H, Shen Y et al. Successful targeting of the NRG1 pathway indicates novel treatment strategy for metastatic cancer. Ann. Oncol. doi: 10.1093/annonc/mdx523 (2017) (Epub ahead of print).Crossref, Google Scholar8 Shin DH, Lee D, Hong DW et al. Oncogenic function and clinical implications of SLC3A2–NRG1 fusion in invasive mucinous adenocarcinoma of the lung. Oncotarget 7(43), 69450–69465 (2016).Crossref, Medline, Google Scholar9 Murayama T, Nakaoku T, Enari M et al. Oncogenic fusion gene CD74–NRG1 confers cancer stem cell-like properties in lung cancer through a IGF2 autocrine/paracrine circuit. Cancer Res. 76(4), 974–983 (2016).Crossref, Medline, CAS, Google Scholar10 Luraghi P, Bigatto V, Cipriano E et al. A molecularly annotated model of patient-derived colon cancer stem-like cells to assess genetic and non-genetic mechanisms of resistance to anti-EGFR therapy. Clin. Cancer. Res. doi: 10.1158/1078-0432.CCR-17-2151 (2017) (Epub ahead of print).Crossref, Medline, Google Scholar11 Yang L, Li Y, Shen E et al. NRG1-dependent activation of HER3 induces primary resistance to trastuzumab in HER2-overexpressing breast cancer cells. Int. J. Oncol. 51(5), 1553–1562 (2017).Crossref, Medline, CAS, Google Scholar12 Xia D, Le LP, Iafrate AJ, Lennerz J. KIF13B–NRG1 gene fusion and KRAS amplification in a case of natural progression of lung cancer. Int. J. Surg. Pathol. 25(3), 238–240 (2017).Crossref, Medline, CAS, Google Scholar13 Muscarella LA, Trombetta D, Fabrizio FP et al. ALK and NRG1 fusions coexist in a patient with signet ring cell lung adenocarcinoma. J. Thorac. Oncol. 12(10), E161–E163 (2017).Crossref, Medline, Google Scholar14 Dhanasekaran SM, Balbin OA, Chen G et al. Transcriptome meta-analysis of lung cancer reveals recurrent aberrations in NRG1 and Hippo pathway genes. Nat. Commun. 5, 5893 (2014).Crossref, Medline, CAS, Google Scholar15 Dong X, Fernandez-Salas E, Li E, Wang S. Elucidation of resistance mechanisms to second-generation ALK inhibitors alectinib and ceritinib in non-small-cell lung cancer cells. Neoplasia 18(3), 162–171 (2016).Crossref, Medline, CAS, Google Scholar16 Kimura M, Endo H, Inoue T et al. Analysis of ERBB ligand-induced resistance mechanism to crizotinib by primary culture of lung adenocarcinoma with EML4–ALK fusion gene. J. Thorac. Oncol. 10(3), 527–530 (2015).Crossref, Medline, CAS, Google Scholar17 Ji-Youn H, Kun Young L, Jin Young K et al. EGFR and HER3 inhibition- A novel therapy for invasive mucinous non-small-cell lung cancer harbouring an NRG1 fusion gene. J. Thorac. Oncol. 12(S1), S669, Abstract P3.02C-006 (2016).Google Scholar18 Gay ND, Wang Y, Beadling C et al. Durable response to afatinib in lung adenocarcinoma harboring NRG1 gene fusions. J. Thorac. Oncol. 12(8), E107–E110 (2017).Crossref, Medline, Google Scholar19 Sun M, Behrens C, Feng L et al. HER family receptor abnormalities in lung cancer brain metastases and corresponding primary tumors. Clin. Cancer Res. 15(15), 4829–4837 (2009).Crossref, Medline, CAS, Google Scholar20 Malm M, Frejd FY, Ståhl S, Löfblom J. Targeting HER3 using mono- and bispecific antibodies or alternative scaffolds. MAbs 8(7), 1195–1209 (2016).Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByCancer prognosis and immune systemThe impact of fusion genes on cancer stem cells and drug resistance7 June 2021 | Molecular and Cellular Biochemistry, Vol. 476, No. 10Neuregulin 1 Gene (NRG1). A Potentially New Targetable Alteration for the Treatment of Lung Cancer9 October 2021 | Cancers, Vol. 13, No. 20Clinicopathologic Features and Response to Therapy of NRG1 Fusion–Driven Lung Cancers: The eNRGy1 Global Multicenter RegistryJournal of Clinical Oncology, Vol. 39, No. 25Methods for actionable gene fusion detection in lung cancer: now and in the futurePasquale Pisapia, Francesco Pepe, Roberta Sgariglia, Mariantonia Nacchio, Gianluca Russo, Gianluca Gragnano, Floriana Conticelli, Maria Salatiello, Caterina De Luca, Ilaria Girolami, Albino Eccher, Antonino Iaccarino, Claudio Bellevicine, Elena Vigliar, Umberto Malapelle & Giancarlo Troncone16 September 2021 | Pharmacogenomics, Vol. 22, No. 13An improved assay for detection of theranostic gene translocations and MET exon 14 skipping in thoracic oncologyLaboratory Investigation, Vol. 101, No. 5Oncogenic driver mutations in non-small cell lung cancer: Past, present and futureWorld Journal of Clinical Oncology, Vol. 12, No. 4Tarloxotinib Is a Hypoxia-Activated Pan-HER Kinase Inhibitor Active Against a Broad Range of HER-Family Oncogenes1 March 2021 | Clinical Cancer Research, Vol. 27, No. 5Predictive biomarkers for molecular pathology in lung cancerPasquale Pisapia, Francesco Pepe, Giancarlo Troncone & Umberto Malapelle3 March 2020 | Biomarkers in Medicine, Vol. 14, No. 4Welcome to Volume 8 of Lung Cancer ManagementJennifer Straiton20 December 2018 | Lung Cancer Management, Vol. 8, No. 1 Vol. 6, No. 4 Follow us on social media for the latest updates Metrics History Received 11 November 2017 Accepted 21 November 2017 Published online 5 January 2018 Published in print December 2017 Information© 2018 Future Medicine LtdKeywordsErbB NRG1 fusionNSCLCFinancial & competing interests disclosureThis work was supported by the Italian Ministry of Health (Ricerca Corrente, RC1703LO41 to LA Muscarella and RC1703ON39 to A Rossi), by the '5x1000' voluntary contributions and by the AIRC/MFAG grant 12983 (to LA Muscarella) and FBNC Grant 2015–2016 to LA Muscarella. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.No writing assistance was utilized in the production of this manuscript.PDF download

Overexpression and/or activation of HER2 confers resistance of cancer cells to chemotherapeutic drugs. NRF2 also gives drug resistance of cancer cells through induction of detoxification and/or drug efflux proteins. Although several upstream effectors of NRF2 overlapped with the downstream molecules of HER2 pathway, no direct link between HER2 and NRF2 has ever been established. Here, we identified that co-expression of a constitutively active HER2 (HER2CA) and NRF2 increased the levels of NRF2 target proteins, HO-1 and MRP5. We also identified HER2CA activated the DNA-binding of NRF2 and the antioxidant response element (ARE)-mediated transcription in an NRF2-dependent manner. In addition, NRF2 and HER2CA cooperatively up-regulated the mRNA expression of various drug-resistant and detoxifying enzymes including GSTA2, GSTP1, CYP3A4, HO-1, MRP1, and MRP5. We also demonstrated that NRF2 binds to HER2 not only in transiently transfected HEK293T cells but also in HER2-amplified breast cancer cells. Functionally, overexpression of HER2CA gave resistance of MCF7 breast cancer cells to either paraquat or doxorubicin. Overexpression of dominant negative NRF2 (DN-NRF2) reduced the HER2CA-induced resistance of MCF7 cells to these agents. Taken together, these results suggest that active HER2 binds and regulates the NRF2-dependent transcriptional activation and induces drug resistance of cancer cells.

: The anthracyclines, doxorubicin and epidoxorubicin, continue to be important drugs for the treatment of breast cancer. Recent studies refocus attention to anthracycline-alkylation and crosslinking of DNA as important toxic events triggering cell death. The long term goals of the proposed research are to establish the mechanism for the crosslinking, to produce new mechanism-based anthracycline derivatives which will be active against resistant breast cancer, and to develop a delivery vehicles for the improved drugs. New derivatives have been synthesized and characterized as the formaldehyde conjugates of doxorubicin and epidoxorubicin, doxoform and epidoxoform, respectively. The following results were obtained during the grant period: I) The crystal structure of epidoxorubicin- alkylated DNA shows the epidoxorubicin virtually crosslinking the DNA at NGC sites. 2) Flow cytometry measurements show drug- formaldehyde conjugates are taken up better by both sensitive and resistant breast cancer cells and retained longer than their clinical counterparts. 3) The nucleus of both sensitive and resistant cancer cells is the primary target for drug-formaldehyde conjugates. 4) Drug-formaldehyde conjugates are more toxic to breast cancer cells than confluent mammary epithelial cells. 5) Sensitive but not resistant breast cancer cells show anthracycline induction of formaldehyde synthesis. 6) Sensitive but not resistant breast cancer cells show measurable formaldehyde levels. 7) Apoptosis assays of drug-treated cells show similar patterns for doxorubicin and doxoform consistent with doxoform being a prodrug to the doxorubicin active metabolite. 8) Epidoxoform shows broad spectrum toxicity to human cancer cells including resistant cancer cells. 9) Epidoxoform can be formulated in DMSO/Cremaphor as a drug delivery vehicle. 10) Conjugation to glutathione is not a resistance mechanism for doxorubicin. 11) Peroxidation of unsaturated lip

Cancer cells switch from highly efficient mitochondrial oxidative phosphorylation (OXPHOS) to low efficiency glycolysis for ATP synthesis even when oxygen is abundant, a phenomenon called the Warburg effect. Because of the higher rates of cell growth and proliferation, cancer cells need more ATP than normal cells of the same tissues. However, the switch is counterintuitive since cancer cells need more ATP but cancer cells prefer to use a low efficiency pathway to produce ATP. How cancer cells secure all their ATP needs is not well understood. On the other hand, extracellular ATP concentration in cancer (intratumoral ATP concentration) are found to be in the range of several hundred μM, ∼10^3-10^4 times higher than those found in normal tissues. Huge differences exist between cancer and normal cells regarding extracellular ATP. Based on these observations, we hypothesized that cancer cells take up ATP from extracellular space to supplement their energy needs. Here we report that in multiple cancer cell lines belonging to multiple cancer types, 0.5-3 mM ATP, within the intratumoral ATP concentration range, elevated intracellular ATP levels by more than 50%. Extracellular ATP also reduced stress and promoted survival of cancer cells that were under OXPHOS and glucose metabolism inhibitions. Furthermore, extracellular ATP increased the viability of cancer cells treated with sunitinib or pazopanib, anticancer drugs working as ATP analogs by competing with ATP for the ATP binding site of targeted tyrosine kinases (TKs). In contrast, extracellular ATP did not affect the activity of paclitaxel, a drug unrelated to ATP. Inhibitor studies revealed that the extracellular ATP-induced intracellular ATP increase shows no dependence on OXPHOS, glycolysis, master ATP regulator AMPK, transcription or translation. These results strongly suggest that not all the increased intracellular ATP is synthesized inside of cancer cells. Fluorescence microscopy study showed that human lung cancer A549 cells, which are KRas oncogene mutation-positive (KRas+), exhibit macropinotcytosis, a type of endocytosis that non-specifically take in extracellular nutritional molecules by “fluid drinking”. Inhibition of macropinocytosis resulted in decrease in dextran take-up and intracellular ATP levels, and alleviated the drug resistance to TKIs in dose-dependent manners. These strongly suggest that macropinocytosis is responsible, at least in part, for transporting extracellular ATP into cancer cells, which led to drug resistance. All these results demonstrate, for the first time, unrecognized roles of extracellular ATP in contributing to the intracellular ATP pool and increase drug resistance in cancer cells and suggest a novel ATP transport mechanism. These findings challenge our traditional view on ATP and shed new lights on ATP homeostasis and ATP-induced drug resistance in cancer cells. Citation Format: Yanrong Qian, Xuan Wang, Yi Liu, Yunsheng Li, Robert A. Colvin, Lingying Tong, Shiyong Wu, Xiaozhuo Chen. Extracellular ATP-induced intracellular ATP concentration elevation mediated by macropinocytosis promotes growth, survival, and drug resistance of cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3364. doi:10.1158/1538-7445.AM2014-3364