Chemotherapy-induced Stress Research Articles

Epithelial and mesenchymal phenotypes determine the dynamics of circulating breast tumor cells in microfluidic capillaries under chemotherapy-induced stress.

Circulating tumor cells (CTCs) with different epithelial and mesenchymal phenotypes play distinct roles in the metastatic cascade. However, the influence of their phenotypic traits and chemotherapy on their transit and retention within capillaries remains unclear. To explore this, we developed a microfluidic device comprising 216 microchannels of different widths from 5 to 16 μm to mimic capillaries. This platform allowed us to study the behaviors of human breast cancer epithelial MCF-7 and mesenchymal MDA-MB-231 cells through microchannels under chemotherapy-induced stress. Our results revealed that when the cell diameter to microchannel width ratio exceeded 1.2, MCF-7 cells exhibited higher transit percentages than MDA-MB-231 cells under a flow rate of 0.13 mm/s. Tamoxifen (250 nM) reduced the transit percentage of MCF-7 cells, whereas 100 nM paclitaxel decreased transit percentages for both cell types. These differential responses were partially due to altered cell stiffness following drug treatments. When cells were entrapped at microchannel entrances, tamoxifen, paclitaxel, and high-flow stress (0.5 mm/s) induced a reduction in mitochondrial membrane potential (MMP) in MCF-7 cells. Tamoxifen treatment also elevated reactive oxygen species (ROS) levels in MCF-7 cells. Conversely, MMP and ROS levels in entrapped MDA-MB-231 cells remained unaffected. Consequently, the viability and proliferation of entrapped MCF-7 cells declined under these chemical and physical stress conditions. Our findings emphasize that phenotypically distinct CTCs may undergo selective filtration and exhibit varied responses to chemotherapy in capillaries, thereby impacting cancer metastasis outcomes. This highlights the importance of considering both cell phenotype and drug response to improve treatment strategies.

PTEN-induced kinase 1 gene single-nucleotide variants as biomarkers in adjuvant chemotherapy for colorectal cancer: a retrospective study

BackgroundFluoropyrimidine-based postoperative adjuvant chemotherapy is globally recommended for high-risk stage II and stage III colon cancer. However, adjuvant chemotherapy is often associated with severe adverse events and is not highly effective in preventing recurrence. Therefore, discovery of novel molecular biomarkers of postoperative adjuvant chemotherapy to identify patients at increased risk of recurrent colorectal cancer is warranted. Autophagy (including mitophagy) is activated under chemotherapy-induced stress and contributes to chemotherapy resistance. Expression of autophagy-related genes and their single-nucleotide polymorphisms are reported to be effective predictors of chemotherapy response in some cancers. Our goal was to evaluate the relationship between single-nucleotide variants of autophagy-related genes and recurrence rates in order to identify novel biomarkers that predict the effect of adjuvant chemotherapy in colorectal cancer. MethodsWe analyzed surgical or biopsy specimens from 84 patients who underwent radical surgery followed by fluoropyrimidine-based adjuvant chemotherapy at Saitama Medical University International Medical Center between January and December 2016. Using targeted enrichment sequencing, we identified single-nucleotide variants and insertions/deletions in 50 genes, including autophagy-related genes, and examined their association with colorectal cancer recurrence rates.ResultsWe detected 560 single-nucleotide variants and insertions/deletions in the target region. The results of Fisher’s exact test indicated that the recurrence rate of colorectal cancer after adjuvant chemotherapy was significantly lower in patients with the single-nucleotide variants (c.1018G > A [p < 0.005] or c.1562A > C [p < 0.01]) of the mitophagy-related gene PTEN-induced kinase 1.ConclusionsThe two single-nucleotide variants of PINK1 gene may be biomarkers of non-recurrence in colorectal cancer patients who received postoperative adjuvant chemotherapy.

203TiP Autophagy as a target for therapy in ovarian cancer: A phase II randomized trial with biomarker correlation (ATOC Trial)

Epithelial ovarian cancer (EOC) is gynecologic cancer with one of the highest mortalities. First-line treatment usually involves cytoreductive surgery and six cycles of platinum and taxane chemotherapy. With this treatment, most (70-80%) patients with stage III/IV disease relapse after one year. Patients who require re-treatment >6 months after previous platinum exposure [platinum sensitive relapsed ovarian cancer (PS-ROC)] are usually treated with a combination containing platinum agent. Autophagy pathways are activated in relapsed ovarian cancer allowing cancer cells to survive chemotherapy-induced stress.

Abstract P1-08-15: Communication between tumor cells and fibroblasts as a prognostic factor of NACT in TNBC

Abstract Introduction. Neoadjuvant chemotherapy (NACT) is the standard of care in aggressive breast cancer, including triple negative breast cancer (TNBC). Cytotoxic drugs specifically target proliferating cells, however, patient outcome is variable. Tumor physiology and response to therapy are orchestrated by an intricate interplay between cancer, stromal and immune cells collectively forming the tumor microenvironment. We have recently uncovered a feedback mechanism of tumor cells and fibroblasts, involving IFNB1 signaling, that supports tumor cells in the recovery from chemotherapy-induced stress. Here, we wanted to assess whether targets of IFNB1 signaling in fibroblasts and tumor cells would qualify as predictive markers of pathological complete response (pCR) after neoadjuvant therapy (NACT) as prognostic markers for the course of the disease. Methods and Patients. RNA-sequencing data from in vitro experiments found the GO-term GO:0051607 ‘defense response to virus’ significantly enriched. Twentyfour genes intersected between differentially expressed genes and the genes of this GO-term. We selected three of the encoded proteins a) interferon induced with helicase C domain 1 IFIH1, b) interferon alpha-inducible protein ISG15, c) 2'-5'-oligoadenylate synthetase OAS1 to test their expression in human specimens of TNBCs after NACT by immunohistochemistry (IHC). None of the respective genes correlated with recurrence free survival when tested in treatment-naïve tumor biopsies (KM Plotter). A prospective consecutively enrolled cohort (2000 - 2021) was available with an overall pCR rate of 46%. pCR was defined by no invasive cancer cell in breast or axilla (ypT0 N0). The median follow-up for iDFS was 36.2 months (6-154) and for OS 39.3 months (6-214).Primary objective was the correlation between IFIH1, ISG15 and OAS1 protein expression in the residual tumor by non-pCR or in the tumor bed by pCR. Second objective was the association of IFIH1, ISG15 and OAS1 protein expression to invasive disease-free survival (iDFS) and overall survival (OS). Results. To date, IHC staining has been established for IFIH1, ISG15 and OAS1. In representative stainings of FFPE tissue samples with pCR we did not detect any positive signal for IFIH1 and ISG15 in stromal cells like fibroblast or lymphocytes. Slight to strong protein expression was detected by non-pCR in cancer cells, stromal cells and tumour infiltrating lymphocytes. In contrast OAS1 was expressed especially strong in lymphocytes by pCR or non-pCR. Cancer cell showed moderate OAS1 expression. IHC analysis and of the entire cohort is in progress including the analyses of the association of these markers to pCR, non-pCR and iDFS/OS. Conclusion. Using samples from our consecutive, multicentre enrolled cohort, an association between the expression of markers of an IFNB1-triggered antiviral response and pCR and survival was demonstrated in patients of the TNBC subgroup. Analysis of the entire cohort is necessary to potentially demonstrate applicability of an interferon-response as predictor of survival. Citation Format: Marcus Bauer, Martina Vetter, Ana Maia, Efstathios Vlachavas, Brigitta Michels, Mireia Berdiel-Acer, Kathleen Schüler, Alessandra Morselli, Manio Skarlatou, Christoph Thomssen, Stefan Wiemann. Communication between tumor cells and fibroblasts as a prognostic factor of NACT in TNBC [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-08-15.

Haploid Kmt2c Deletions Enhance Hematopoietic Stem Cell Mobilization in Response to Granulocyte-Colony Stimulating Factor

KMT2C is one of several tumor suppressor genes deleted in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) as part of larger chromosome 7q deletions. These deletions are particularly common in therapy-related MDS/AML, raising the question of whether loss of one or more 7q genes conveys a selective advantage to hematopoietic stem cells (HSCs) in the setting of chemotherapy-induced stress. We recently showed that haploid Kmt2c deletions do indeed enhance HSC self-renewal capacity. However, the deletions do not drive HSCs into cycle; instead, a proliferative stress such as chemotherapy is required to create a context in which Kmt2c deletion convey a selective advantage. We have also identified a mechanism, to explain this phenotype. Kmt2c encodes MLL3, a SET domain protein that binds enhancers and facilitates transcription. We have shown that Kmt2c/MLL3 deletions impairs enhancer recruitment during HSC differentiation, therefore blunting HSC commitment.Our findings suggest that acquired or pre-existing 7q (KMT2C) deletions may select for HSCs that could give rise to MDS/AML in the setting of autologous-transplantation. Granulocyte-colony stimulating factor (G-CSF) is a cytokine that is often used to expedite neutrophil recovery after chemotherapy and to mobilize HSCs for collection and transplantation. We considered the possibility that 7q deletions, and KMT2C deletions in particular, may promote disproportionate mobilization of the mutant HSCs in response to G-CSF. To test this, we treated Kmt2c f/f; Vav1-Cre and Kmt2c f/+; Vav1-Cre mice with G-CSF, and we assessed HSC mobilization to the spleen and bone marrow. A far greater proportion of HSCs with heterozygous and homozygous Kmt2c deletions mobilized in response to G-CSF, relative to wild type HSCs. Kmt2c deletion also enhanced colony forming unit frequency in the peripheral blood after G-CSF treatment. Total body HSC numbers did not change in the body after G-CSF treatment on any genetic background, indicating that Kmt2c deletions enhanced HSC mobilization in response to G-CSF rather than self-renewal.To more faithfully recapitulate clinical conditions, we used Fgd5-CreER to delete a single Kmt2c allele in only a minority of HSCs. We then tested whether the mutant HSCs mobilized more efficiently than wild type HSCs. Surprisingly, Kmt2c deletions did not enhance HSC mobilization in this context. This raised the question of whether Kmt2c deletion in a non-HSC population could promote HSC mobilization in the Kmt2c f/+; Vav1-Cre mice. Indeed, analysis of mice chimeric for wild type and Kmt2c f/+; Vav1-Cre bone marrow suggested that Kmt2c non-cell autonomously regulates HSC mobilization. Finally, Kmt2c deletions did not enhance mobilization following exposure to plerixafor, a CXCR4 antagonist that acts directly on HSCs to promote mobilization rather than indirectly via monocyte populations, as occurs with G-CSF. Additional studies are needed to elucidate the mechanism by which Kmt2c non-cell autonomously regulates HSC mobilization. Our findings provide reassurance that, in a clinical setting, rare KMT2C-mutant HSCs will not disproportionately mobilize prior to apheresis. Furthermore, the data suggest that transient inhibition of MLL3, or its targets, may enhance HSC mobilization and negate selective advantages that 7q-deleted HSCs may acquire after chemotherapy treatment. DisclosuresNo relevant conflicts of interest to declare.

Arachidonic acid drives adaptive responses to chemotherapy-induced stress in malignant mesothelioma

BackgroundHigh resistance to therapy and poor prognosis characterizes malignant pleural mesothelioma (MPM). In fact, the current lines of treatment, based on platinum and pemetrexed, have limited impact on the survival of MPM patients. Adaptive response to therapy-induced stress involves complex rearrangements of the MPM secretome, mediated by the acquisition of a senescence-associated-secretory-phenotype (SASP). This fuels the emergence of chemoresistant cell subpopulations, with specific gene expression traits and protumorigenic features. The SASP-driven rearrangement of MPM secretome takes days to weeks to occur. Thus, we have searched for early mediators of such adaptive process and focused on metabolites differentially released in mesothelioma vs mesothelial cell culture media, after treatment with pemetrexed.MethodsMass spectrometry-based (LC/MS and GC/MS) identification of extracellular metabolites and unbiased statistical analysis were performed on the spent media of mesothelial and mesothelioma cell lines, at steady state and after a pulse with pharmacologically relevant doses of the drug. ELISA based evaluation of arachidonic acid (AA) levels and enzyme inhibition assays were used to explore the role of cPLA2 in AA release and that of LOX/COX-mediated processing of AA. QRT-PCR, flow cytometry analysis of ALDH expressing cells and 3D spheroid growth assays were employed to assess the role of AA at mediating chemoresistance features of MPM. ELISA based detection of p65 and IkBalpha were used to interrogate the NFkB pathway activation in AA-treated cells.ResultsWe first validated what is known or expected from the mechanism of action of the antifolate. Further, we found increased levels of PUFAs and, more specifically, arachidonic acid (AA), in the transformed cell lines treated with pemetrexed. We showed that pharmacologically relevant doses of AA tightly recapitulated the rearrangement of cell subpopulations and the gene expression changes happening in pemetrexed -treated cultures and related to chemoresistance. Further, we showed that release of AA following pemetrexed treatment was due to cPLA2 and that AA signaling impinged on NFkB activation and largely affected anchorage-independent, 3D growth and the resistance of the MPM 3D cultures to the drug.ConclusionsAA is an early mediator of the adaptive response to pem in chemoresistant MPM and, possibly, other malignancies.

A combined treatment regimen of MGMT-modified \u03b3\u03b4 T cells and temozolomide chemotherapy is effective against primary high grade gliomas

Chemotherapeutic drugs such as the alkylating agent Temozolomide (TMZ), in addition to reducing tumor mass, can also sensitize tumors to immune recognition by transient upregulation of multiple stress induced NKG2D ligands (NKG2DL). However, the potential for an effective response by innate lymphocyte effectors such as NK and γδ T cells that recognize NKG2DL is limited by the drug’s concomitant lymphodepleting effects. We have previously shown that modification of γδ T cells with a methylguanine DNA methyltransferase (MGMT) transgene confers TMZ resistance via production of O6-alkylguanine DNA alkyltransferase (AGT) thereby enabling γδ T cell function in therapeutic concentrations of TMZ. In this study, we tested this strategy which we have termed Drug Resistant Immunotherapy (DRI) to examine whether combination therapy of TMZ and MGMT-modified γδ T cells could improve survival outcomes in four human/mouse xenograft models of primary and refractory GBM. Our results confirm that DRI leverages the innate response of γδ T cells to chemotherapy-induced stress associated antigen expression and achieves synergies that are significantly greater than either individual approach.

Systems level profiling of chemotherapy-induced stress resolution in cancer cells reveals druggable trade-offs.

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.

Group 2 innate lymphoid cells support hematopoietic recovery under stress conditions.

The cell-cycle status of hematopoietic stem and progenitor cells (HSPCs) becomes activated following chemotherapy-induced stress, promoting bone marrow (BM) regeneration; however, the underlying molecular mechanism remains elusive. Here we show that BM-resident group 2 innate lymphoid cells (ILC2s) support the recovery of HSPCs from 5-fluorouracil (5-FU)-induced stress by secreting granulocyte-macrophage colony-stimulating factor (GM-CSF). Mechanistically, IL-33 released from chemo-sensitive B cell progenitors activates MyD88-mediated secretion of GM-CSF in ILC2, suggesting the existence of a B cell-ILC2 axis for maintaining hematopoietic homeostasis. GM-CSF knockout mice treated with 5-FU showed severe loss of myeloid lineage cells, causing lethality, which was rescued by transferring BM ILC2s from wild-type mice. Further, the adoptive transfer of ILC2s to 5-FU-treated mice accelerates hematopoietic recovery, while the reduction of ILC2s results in the opposite effect. Thus, ILC2s may function by "sensing" the damaged BM spaces and subsequently support hematopoietic recovery under stress conditions.

Abstract PO-051: Metabolic alterations reveal epigenetic vulnerabilities in breast cancer

Abstract Tumor progression and therapeutic resistance in cancer are characterized by changes in cell identity, which is regulated in part by the epigenetic landscape of the cells. Alterations in metabolic profiles can contribute to changes in cell fate during tumor progression through remodelling of chromatin landscapes. We show that chemoresistance in triple-negative breast cancer is characterized by altered cellular metabolism, impacting the levels of metabolites required for DNA and histone methylation. Using models of chemotherapy resistance breast cancer, we have identified metabolic and epigenetic dependencies specific to drug-resistant breast cancer cells. We show that the remodelling of epigenetic states upon chemotherapy-induced metabolic stress can contribute to drug resistance in breast cancer cells by enabling the maintenance of transposable element repression and preventing the induction of cytotoxic stress-induced viral mimicry. While these metabolic and epigenetic changes contribute to the enhanced tumorigenic fitness of chemo resistant breast cancer, they reveal epigenetic vulnerabilities that can be therapeutically targeted in drug-resistant tumors. Citation Format: Genevieve Deblois. Metabolic alterations reveal epigenetic vulnerabilities in breast cancer [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-051.

Overcoming Wnt-β-catenin dependent anticancer therapy resistance in leukaemia stem cells.

Leukaemia stem cells (LSCs) underlie cancer therapy resistance but targeting these cells remains difficult. The Wnt-β-catenin and PI3K-Akt pathways cooperate to promote tumorigenesis and resistance to therapy. In a mouse model in which both pathways are activated in stem and progenitor cells, LSCs expanded under chemotherapy-induced stress. Since Akt can activate β-catenin, inhibiting this interaction might target therapy-resistant LSCs. High-throughput screening identified doxorubicin (DXR) as an inhibitor of the Akt-β-catenin interaction at low doses. Here we repurposed DXR as a targeted inhibitor rather than a broadly cytotoxic chemotherapy. Targeted DXR reduced Akt-activated β-catenin levels in chemoresistant LSCs and reduced LSC tumorigenic activity. Mechanistically, β-catenin binds multiple immune-checkpoint gene loci, and targeted DXR treatment inhibited expression of multiple immune checkpoints specifically in LSCs, including PD-L1, TIM3 and CD24. Overall, LSCs exhibit distinct properties of immune resistance that are reduced by inhibiting Akt-activated β-catenin. These findings suggest a strategy for overcoming cancer therapy resistance and immune escape.

NKG2D Ligand Shedding in Response to Stress: Role of ADAM10.

NKG2D is an activating receptor expressed by NK cells and some subsets of T cells and represents a major recognition receptor for detection and elimination of cancer cells. The ligands of NKG2D are stress-induced self-proteins that can be secreted as soluble molecules by protease-mediated cleavage. The release of NKG2D ligands in the extracellular milieu is considered a mode of finely controlling their surface expression levels and represents a relevant immune evasion mechanism employed by cancer cells to elude NKG2D-mediated immune surveillance. A disintegrin and metalloproteinase 10 (ADAM10), a catalytically active member of the ADAM family of proteases, is involved in the cleavage of some NKG2D ligands in various types of cancer cells either in steady state conditions and in response to an ample variety of stress stimuli. Appealing immunotherapeutic strategies devoted to promoting NK cell-mediated recognition and elimination of cancer cells are based on the upregulation of NK cell activating ligands. In particular, activation of DNA damage response (DDR) and the induction of cellular senescence by chemotherapeutic agents are associated with increased expression of NKG2D ligands on cancer cell surface. Herein, we will review advances on the protease-mediated cleavage of NKG2D ligands in response to chemotherapy-induced stress focusing on: (i) the role played by ADAM10 in this process and (ii) the implications of NKG2D ligand shedding in the course of cancer therapy and in senescent cells.

Kmt2c Limits the Self-Renewal Capacity of Multiply Divided HSCs By Promoting Sensitivity to Interleukin-1

Under normal homeostatic conditions, adult hematopoietic stem cells (HSCs) are usually quiescent. Hematopoietic stress, such as blood loss, infection, inflammation, or chemotherapy, can drive HSCs into cycle. When adult HSCs divide multiple times, they lose self-renewal capacity. Inflammatory cytokines, such as interleukin-1 (IL-1), can accelerate the loss of HSC self-renewal capacity by activating PU.1 and promoting myeloid commitment. This raises the question of whether intrinsic tumor suppressor genes can modulate sensitivity to inflammatory cytokines, and whether loss of these tumor suppressors can allow HSCs to evade commitment programs that would otherwise limit self-renewal capacity. The KMT2C tumor suppressor is located on chromosome 7q within a region that is frequently deleted in myelodsplastic syndrome (MDS) and therapy-related acute myeloid leukemia (AML). It encodes MLL3, a histone methyltransferase that activates enhancer elements and promotes transcription. Haploid KMT2C deletion has previously been shown to activate self-renewal programs and accelerate AML formation. This raised the question of whether KMT2C/MLL3 regulates normal HSC self-renewal and whether KMT2C deletion conveys a selective advantage to HSCs in contexts that would otherwise deplete the HSC pool. By protecting HSCs from exhaustion, KMT2C deletions may indirectly facilitate 7q-deficient MDS/AML. To understand whether and how Kmt2c regulates HSC self-renewal, we developed novel germline and conditional Kmt2c knockout mouse alleles. Mono- and bi-allelic Kmt2c deletions led to a modest increase in adult HSC numbers and a significant reduction in committed hematopoietic progenitors (HPCs). Kmt2c deletions markedly enhanced HSC self-renewal capacity, but HSC proliferation rates were not altered. To mimic conditions that lead to therapy-related AML, we deleted a single Kmt2c allele in a minority of HSCs. We then tested whether the Kmt2c-deleted HSC population expanded, relative to wild type HSCs, under native and stressed conditions. Under native conditions, the percentage of Kmt2c-heterozygous HSCs remained stable. However, after several cycles of chemotherapy, the Kmt2c mutant HSCs predominated within the marrow. In mechanistic studies, RNA-sequencing showed that Kmt2c-deficient HSCs expressed genes associated with innate immune signaling, including the receptor for interleukin-1 (IL1R), at lower levels than wild type HSCs. This suggested that Kmt2c mutations might sustain self-renewal capacity in multiply divided HSCs by dampening IL-1 driven myeloid commitment. In support of this hypothesis, Kmt2c-deficient HSCs retained multilineage potential when they were cultured with IL-1, and they failed to activate JNK and p38 upon exposure to IL-1. Altogether, our data suggest a mechanism to explain how KMT2C deletions, in the context of larger 7q deletions, may promote therapy related MDS/AML. When HSCs acquire a KMT2C deletion, they can then escape IL-1-mediated exhaustion when they are driven into cycle by chemotherapy or other stressors. In lieu of chemotherapy-induced stress, the same clones may remain relatively indolent. Disclosures No relevant conflicts of interest to declare.

Cancer Exosomes as Conveyors of Stress-Induced Molecules: New Players in the Modulation of NK Cell Response.

Natural killer (NK) cells are innate lymphoid cells that play a pivotal role in tumor surveillance. Exosomes are nanovesicles released into the extracellular environment via the endosomal vesicle pathway and represent an important mode of intercellular communication. The ability of anticancer chemotherapy to enhance the immunogenic potential of malignant cells mainly relies on the establishment of the immunogenic cell death (ICD) and the release of damage-associated molecular patterns (DAMPs). Moreover, the activation of the DNA damage response (DDR) and the induction of senescence represent two crucial modalities aimed at promoting the clearance of drug-treated tumor cells by NK cells. Emerging evidence has shown that stress stimuli provoke an increased release of exosome secretion. Remarkably, tumor-derived exosomes (Tex) produced in response to stress carry distinct type of DAMPs that activate innate immune cell populations. Moreover, stress-induced ligands for the activating receptor NKG2D are transported by this class of nanovesicles. Here, we will discuss how Tex interact with NK cells and provide insight into their potential role in response to chemotherapy-induced stress stimuli. The capability of some “danger signals” carried by exosomes that indirectly affect the NK cell activity in the tumor microenvironment will be also addressed.

P1253Clinical features and outcomes of patients with chemotherapy-induced Takotsubo stress cardiomyopathy

P1253Clinical features and outcomes of patients with chemotherapy-induced Takotsubo stress cardiomyopathy

Modeling chemotherapy-induced stress to identify rational combination therapies in the DNA damage response pathway.

Cells respond to DNA damage by activating complex signaling networks that decide cell fate, promoting not only DNA damage repair and survival but also cell death. We have developed a multiscale computational model that quantitatively links chemotherapy-induced DNA damage response signaling to cell fate. The computational model was trained and calibrated on extensive data from U2OS osteosarcoma cells, including the cell cycle distribution of the initial cell population, signaling data measured by Western blotting, and cell fate data in response to chemotherapy treatment measured by time-lapse microscopy. The resulting mechanistic model predicted the cellular responses to chemotherapy alone and in combination with targeted inhibitors of the DNA damage response pathway, which we confirmed experimentally. Computational models such as the one presented here can be used to understand the molecular basis that defines the complex interplay between cell survival and cell death and to rationally identify chemotherapy-potentiating drug combinations.

28 Stress signalling in breast cancer cells induces matrix components of stem cell niches that promote chemotherapy-resistant metastasis

IntroductionCancer progression to metastasis remains a major clinical challenge and is commonly associated with resistance to prevailing therapies such as chemotherapy. Exposure to cytotoxic therapies may cause extensive stress responses in cancer cells. The c-Jun N-terminal kinase (JNK) pathway is induced by stress and can be activated by chemotherapy. However, the cellular outcome of JNK signalling in metastatic cancer is still paradoxical and poorly understood.Material and methodsIn our experiments, we utilised 3D culture systems of primary breast cancer cells, transcriptomic screens and xenograft mouse models of metastasis. We validated our findings using annotated clinically derived material, such as pleural effusions, patient-derived gene expression profiles or tissue microarrays.Results and discussionsWe show that high JNK stress signalling in human breast cancer samples predicts poor overall survival and is enriched in dissected nodules of lung metastases compared to patient-matched primary tumours. In experimental models, we show that JNK signalling, which is active in a subpopulation of cancer cells, induces ECM, wound healing and stem cell network that promotes lung metastasis in breast cancer. We find that two ECM proteins of stem cell niches, osteopontin (SPP1) and tenascin C (TNC), play a major role in the JNK-induced network and are direct targets of the c-Jun transcription factor, downstream of JNK signalling. Both SPP1 and TNC act as essential mediators of metastasis to the lungs. Exposure to multiple chemotherapies further exploits this JNK-mediated axis to confer treatment resistance. Importantly, JNK inhibition, or disruption of SPP1 or TNC expression, sensitises experimental mammary tumours and metastases to chemotherapy and may therefore represent a promising new treatment strategy against metastatic breast cancer.ConclusionOur work provides insight into how cancer cells can benefit from chemotherapy-induced stress signals that counteract cytotoxic responses to the therapy. The identification of SPP1 and TNC as mediators of therapy resistance underscores the significance of the ECM as a valuable source of cancer targets. Moreover, our results provide rationale for using JNK inhibition to repress simultaneously the expression of SPP1 and TNC, and thus sensitise metastatic breast cancer to chemotherapy.

The cytoprotective protein clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and promotes gastric cancer cell survival

The cytoprotective protein clusterin is often dysregulated during tumorigenesis, and in the stomach, upregulation of clusterin marks emergence of the oxyntic atrophy (loss of acid-producing parietal cells)-associated spasmolytic polypeptide-expressing metaplasia (SPEM). The hormone gastrin is important for normal function and maturation of the gastric oxyntic mucosa and hypergastrinemia might be involved in gastric carcinogenesis. Gastrin induces expression of clusterin in adenocarcinoma cells. In the present study, we examined the expression patterns and gastrin-mediated regulation of clusterin in gastric tissue from: humans; rats treated with proton pump (H+/K+-ATPase) inhibitors and/or a gastrin receptor (CCK2R) antagonist; H+/K+-ATPase β-subunit knockout (H/K-β KO) mice; and Mongolian gerbils infected with Helicobacter pylori and given a CCK2R antagonist. Biological function of secretory clusterin was studied in human gastric cancer cells. Clusterin was highly expressed in neuroendocrine cells in normal oxyntic mucosa of humans and rodents. In response to hypergastrinemia, expression of clusterin increased significantly and its localization shifted to basal groups of proliferative cells in the mucous neck cell-chief cell lineage in all animal models. That shift was partially inhibited by antagonizing the CCK2R in rats and gerbils. The oxyntic mucosa of H/K-β KO mice contained areas with clusterin-positive mucous cells resembling SPEM. In gastric adenocarcinomas, clusterin mRNA expression was higher in diffuse tumors containing signet ring cells compared with diffuse tumors without signet ring cells, and clusterin seemed to be secreted by tumor cells. In gastric cancer cell lines, gastrin increased secretion of clusterin, and both gastrin and secretory clusterin promoted survival after starvation- and chemotherapy-induced stress. Overall, our results indicate that clusterin is overexpressed in hypergastrinemic rodent models of oxyntic preneoplasia and stimulates gastric cancer cell survival.

Linking genomic reorganization to tumor initiation via the giant cell cycle.

To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse.

Inhibition of the colony-stimulating-factor-1 receptor affects the resistance of lung cancer cells to cisplatin.

In the present work we show that multiple lung cancer cell lines contain cisplatin resistant cell subpopulations expressing the Colony-Stimulating-Factor-Receptor-1 (CSF-1R) and surviving chemotherapy-induced stress. By exploiting siRNA-mediated knock down in vitro and the use of an investigational CSF-1R TKI (JNJ-40346527) in vitro and in vivo, we show that expression and function of the receptor are required for the clonogenicity and chemoresistance of the cell lines. Thus, inhibition of the kinase activity of the receptor reduced the levels of EMT-associated genes, stem cell markers and chemoresistance genes. Additionally, the number of high aldehyde dehydrogenase (ALDH) expressing cells was reduced, consequent to the lack of cisplatin-induced increase of ALDH isoforms. This affected the collective chemoresistance of the treated cultures. Treatment of tumor bearing mice with JNJ-40346527, at pharmacologically relevant doses, produced strong chemo-sensitizing effects in vivo. These anticancer effects correlated with a reduced number of CSF-1Rpos cells, in tumors excised from the treated mice. Depletion of the CD45pos cells within the treated tumors did not, apparently, play a major role in mediating the therapeutic response to the TKI. Thus, lung cancer cells express a functional CSF-1 and CSF-1R duo which mediates pro-tumorigenic effects in vivo and in vitro and can be targeted in a therapeutically relevant way. These observations complement the already known role for the CSF-1R at mediating the pro-tumorigenic properties of tumor-infiltrating immune components.