Met-1 Cells Research Articles

Fabrication of silver nanoparticles immobilized on magnetic lignosulfonate: Evaluation of its catalytic activity in the N-acetylation reactions and investigation of its anti-cutaneous squamous cell carcinoma effects

Due to the non-optimal response of most types of cancer to various treatment methods and their rapid progress, research continues in the field of producing drugs with less toxicity and greater efficiency. There are many nanocomposites with diverse biological activities that include part of anticancer drugs in new pharmacological science. The present investigation describes a green procedure for the in situ support of Ag nanoparticles (NPs) over sodium lignosulfonate (NaLS) modified magnetic nanoparticles (Fe3O4@NaLS/Ag) and its subsequent biological and chemical performance. FT-IR, TEM, FE-SEM, EDS, ICP, VSM and XRD techniques were used to characterize the synthesized Fe3O4@NaLS/Ag. The catalytic efficacy of the desired composite was applied in the N-acetylation of various amines in the presence of Ac2O under solvent-free conditions. The Fe3O4@NaLS/Ag catalyst was recovered by an external magnet and reused for nine runs without a significant decrease in the activity. The cytotoxic and anti-cutaneous squamous cell carcinoma potentials of biologically synthesized Fe3O4@NaLS/Ag nanocomposite against PM1 and MET1 cells were determined. The anti-cutaneous squamous cell carcinoma properties of the Fe3O4@NaLS/Ag nanocomposite could significantly remove PM1 and MET1 cells. The IC50 of Fe3O4@NaLS/Ag nanocomposite was 288 and 270 μg/mL against PM1 and MET1 cells, respectively. Also, Fe3O4@NaLS/Ag nanocomposite presented a high antioxidant potential according to the IC50 value. According to the above results, the recent nanocomposite can be used in treating cutaneous squamous cell carcinoma after doing clinical trial studies.

Phototoxic Potential of Different DNA Intercalators for Skin Cancer Therapy: In Vitro Screening.

Photodynamic therapy is a minimally invasive procedure used in the treatment of several diseases, including some types of cancer. It is based on photosensitizer molecules, which, in the presence of oxygen and light, lead to the formation of reactive oxygen species (ROS) and consequent cell death. The selection of the photosensitizer molecule is important for the therapy efficiency; therefore, many molecules such as dyes, natural products and metallic complexes have been investigated regarding their photosensitizing potential. In this work, the phototoxic potential of the DNA-intercalating molecules-the dyes methylene blue (MB), acridine orange (AO) and gentian violet (GV); the natural products curcumin (CUR), quercetin (QT) and epigallocatechin gallate (EGCG); and the chelating compounds neocuproine (NEO), 1,10-phenanthroline (PHE) and 2,2'-bipyridyl (BIPY)-were analyzed. The cytotoxicity of these chemicals was tested in vitro in non-cancer keratinocytes (HaCaT) and squamous cell carcinoma (MET1) cell lines. A phototoxicity assay and the detection of intracellular ROS were performed in MET1 cells. Results revealed that the IC50 values of the dyes and curcumin in MET1 cells were lower than 30 µM, while the values for the natural products QT and EGCG and the chelating agents BIPY and PHE were higher than 100 µM. The IC50 of MB and AO was greatly affected by irradiation when submitted to 640 nm and 457 nm light sources, respectively. ROS detection was more evident for cells treated with AO at low concentrations. In studies with the melanoma cell line WM983b, cells were more resistant to MB and AO and presented slightly higher IC50 values, in line with the results of the phototoxicity assays. This study reveals that many molecules can act as photosensitizers, but the effect depends on the cell line and the concentration of the chemical. Finally, significant photosensitizing activity of acridine orange at low concentrations and moderate light doses was demonstrated.

Kindlin-1 regulates IL-6 secretion and modulates the immune environment in breast cancer models.

The adhesion protein Kindlin-1 is over-expressed in breast cancer where it is associated with metastasis-free survival; however, the mechanisms involved are poorly understood. Here, we report that Kindlin-1 promotes anti-tumor immune evasion in mouse models of breast cancer. Deletion of Kindlin-1 in Met-1 mammary tumor cells led to tumor regression following injection into immunocompetent hosts. This was associated with a reduction in tumor infiltrating Tregs. Similar changes in T cell populations were seen following depletion of Kindlin-1 in the polyomavirus middle T antigen (PyV MT)-driven mouse model of spontaneous mammary tumorigenesis. There was a significant increase in IL-6 secretion from Met-1 cells when Kindlin-1 was depleted and conditioned media from Kindlin-1-depleted cells led to a decrease in the ability of Tregs to suppress the proliferation of CD8+ T cells, which was dependent on IL-6. In addition, deletion of tumor-derived IL-6 in the Kindlin-1-depleted tumors reversed the reduction of tumor-infiltrating Tregs. Overall, these data identify a novel function for Kindlin-1 in regulation of anti-tumor immunity, and that Kindlin-1 dependent cytokine secretion can impact the tumor immune environment.

Abstract 1773: Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology

Abstract Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype for which chemotherapy remains a part of standard treatment. Although pathologic complete response (pCR) after neoadjuvant chemotherapy, including paclitaxel (PTX), is associated with good outcomes, 50-60% of TNBC patients do not experience pCR and suffer poor long-term outcomes, often due to chemotherapy resistance. Identifying and analyzing the tumor cells responsible for chemotherapy resistance will lead to improved treatment strategies. We developed a model of TNBC resistance to neoadjuvant PTX using Met1 murine mammary carcinoma cells. To study clonal dynamics in response to PTX we developed a barcoding method, SunCatcher. First, 31 single-cell derived clonal populations were generated from parental Met1 cells. Each clone was infected with a lentiviral vector containing a unique DNA barcode detectable by qPCR. All barcoded clones (BCs) were mixed in equivalent numbers to generate a BC pool that we confirmed captures the parental Met1 heterogeneity and tumor growth kinetics. First, PTX responses were tested in vitro. The BC pool had an IC50 of ~100 nM, while individual BCs ranged in IC50 from 5 nM to 25 µM. We maintained the BC pool in 100 nM PTX for 39 days (termed long-term PTX; LTP), at which point it became clonal for BC25, suggesting that BC25 (IC50 of 100 nM) is uniquely PTX resistant, despite being the most proliferative and Zeb1hi/EpCAMlow clone in vitro. BC25 was not detected in the control-treated BC pool at 39 days. Next, the BC pool was orthotopically injected into FVB/NJ mice and treated the mice with 20 mg/kg PTX on days 7, 11, and 15. At the day 18 experimental endpoint, tumor volume significantly decreased by 56% in response to PTX compared to control. BC25 composition increased from 7.7% (controls) to 15% (PTX treated) within the tumors at endpoint. We also injected BC25 and LTP cells alone and administered the same PTX regimen once tumors reached 50 mm3. Both BC25 and LTP tumors were unresponsive to PTX and had a longer latency period (35d) than BC pool (10d). We performed a drug screen of 2313 compounds spanning FDA-approved cancer therapeutics on the LTP cells to identify compounds to target the PTX-resistant clone. HDAC inhibitors were the most potent class of hits and one, Panobinostat, killed LTP cells with an IC50 of 4.3 nM. Utilizing SunCatcher, we identified a unique PTX-resistant TNBC subclone that represents residual disease associated with poor long-term outcome. Typical in vivo experiments would have reached ethical endpoint before BC25 had a chance to grow, given its long latency, therefore this PTX-resistant clone would not have been identified. This is an important finding because therapeutic resistance can emerge after a protracted period, longer than typical pre-clinical experiments. Further work will explore mechanisms of resistance and the potential for combination therapies to prevent recurrent disease. Citation Format: Milos Spasic, Qiuchen Guo, Adam Maynard, Gregory Goreczny, Adrienne Waks, Sara Tolaney, Elizabeth Mittendorf, Sandra McAllister. Overcoming paclitaxel resistance in triple-negative breast cancer using a novel barcoding technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1773.

MicroRNA-200c restoration reveals a cytokine profile to enhance M1 macrophage polarization in breast cancer

Many immune suppressive mechanisms utilized by triple negative breast cancer (TNBC) are regulated by oncogenic epithelial-to-mesenchymal transition (EMT). How TNBC EMT impacts innate immune cells is not fully understood. To determine how TNBC suppresses antitumor macrophages, we used microRNA-200c (miR-200c), a powerful repressor of EMT, to drive mesenchymal-like mouse mammary carcinoma and human TNBC cells toward a more epithelial state. MiR-200c restoration significantly decreased growth of mouse mammary carcinoma Met-1 cells in culture and in vivo. Cytokine profiling of Met-1 and human BT549 cells revealed that miR-200c upregulated cytokines, such as granulocyte-macrophage colony-stimulating factor (GM-CSF), promoted M1 antitumor macrophage polarization. Cytokines upregulated by miR-200c correlated with an epithelial gene signature and M1 macrophage polarization in BC patients and predicted a more favorable overall survival for TNBC patients. Our findings demonstrate that immunogenic cytokines (e.g., GM-CSF) are suppressed in aggressive TNBC, warranting further investigation of cytokine-based therapies to limit disease recurrence.

Abstract 2024: Obesity induces breast cancer cell invasion through stromal secretion of IGF-1

Abstract Obese women diagnosed with breast cancer have an increased risk for metastasis, and the underlying mechanisms are not well established. Within the mammary gland, adipose stromal cells (ASCs) are a group of heterogeneous cells with the capacity to differentiate along multiple mesenchymal lineages. ASCs are recruited and activated within tumors, but it is unknown how obesity may influence ASCs in both normal and cancerous tissue. To study the effects of obesity on ASCs, we isolated the stromal vascular fraction from the mammary glands of mice fed either a control diet (CD) or high fat diet (HFD) to induce obesity and plated the cells to select for adherent growth. We observed that obesity increased ASCs proliferation, decreased differentiation potential, and upregulated expression of α-smooth muscle actin, a marker of activated fibroblasts, compared to ASCs from lean mice. To determine how ASCs from obese mice impacted tumor growth, we mixed ASCs isolated from CD- or HFD-fed mice with mammary tumor cells, Met-1 cells, and transplanted them into the mammary glands of lean FVB female mice. Tumors cells mixed with ASCs from obese mice grew significantly larger tumors and had increased invasion into surrounding adipose tissue than tumor cells mixed with control ASCs. To study the effects of factors secreted by ASCs on tumor cell behavior, we collected conditioned media (CM) from lean and obese ASCs and treated Met-1 cells. Compared with lean ASCs, obese ASCs expressed higher levels of insulin-like growth factor-1 (IGF-1), which has been implicated in tumor cell invasion. Met-1 cells treated with recombinant IGF-1 were more invasive than vehicle-treated cells. In addition, Met-1 cells treated with obese ASCs CM supplemented with an IGF-1 neutralizing antibody had significantly reduced invasion, compared to controls. Weight loss induced in obese mice significantly decreased expression of IGF-1 from ASCs and reduced the ability of the ASCs to induce an invasive phenotype. Together, these results suggest that obesity enhances local invasion of breast cancer cells through increased expression of IGF-1 by mammary ASCs, and weight loss may reverse this tumor-promoting phenotype. Citation Format: Lauren Hillers, Joseph D'Amato, Tamara Chamberlin, Gretchen Paderta, Lisa Arendt. Obesity induces breast cancer cell invasion through stromal secretion of IGF-1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2024.

Abstract 5152: EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C

Abstract Breast cancer is a devastating disease that claims around 50 million lives per year worldwide, and almost all of these deaths result from metastatic disease rather than from primary tumor burden. Recent studies clearly demonstrate that there is significant intratumor heterogeneity in breast cancer, and that this heterogeneity contributes to malignant progression. Our laboratories previously demonstrated that breast cancer cells that had undergone an oncogenic epithelial-to-mesenchymal transition (EMT) could increase metastasis of neighboring cells via activation of non-canonical GLI transcription factor activity in a paracrine manner that is dependent on Six1 expression in the EMT cells. However, the mechanism by which these Six1-expressing EMT cells activate GLI signaling thereby imparting aggressive properties on neighboring cells remained unknown. Herein we describe the novel discovery that VEGF-C, which is transcriptionally upregulated by Six1, mediates paracrine non-canonical activation of GLI and resultant enhancement of metastatic properties in cells that do not express Six1. Our data demonstrate that VEGF-C is upregulated in HMLER-Snail1 cells, MCF7-Six1 cells and in Met-1 cells endogenously expressing Six1, in a manner that depends on Six1 expression, and that VEGF-C is secreted into the conditioned media (CM) of these cells. Inhibition of VEGF-C in the HMLER-Snail1, MCF7-Six1, and the Met-1 models abrogates paracrine GLI activation and attenuates non-cell autonomous induction of proliferation, migration, and invasion in vitro. In vivo, we show that cells expressing Six1 can enhance the growth and metastasis of those not expressing Six1, and studies will be discussed that examine whether VEGF-C inhibition in Six1 expressing cells disrupts the crosstalk and inhibits non-cell autonomous induction of metastasis by Six1. Finally, by interrogating the TCGA dataset we find that VEGF-C and GLI1 expression significantly positively correlate in human breast cancer encompassing all molecular subtypes. Taken together, these data suggest that VEGF-C secretion may be a novel and conserved paracrine means by which EMT cells activate GLI in neighboring tumor cells that do not express these EMT-inducing transcription factors (TF), ultimately enhancing overall metastasis of heterogenous breast tumors. Citation Format: Deguang Kong, Deepika Neelakantan, Hengbo Zhou, Michael T. Lewis, Heide L. Ford. EMT cells increase the metastatic potential of neighboring carcinoma cells via non-canonical activation of GLI signaling through secretion of VEGF-C [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5152.

Abstract P2-01-04: Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression

Abstract Background: Epithelial-Mesenchymal Transition (EMT) is a developmental process co-opted by carcinomas to facilitate metastasis. Triple negative breast cancer (TNBC), which has often undergone at least partial EMT, has a poor prognosis due to frequent and rapid recurrence as metastatic disease when compared to other breast cancer subtypes. To identify drivers of TNBC progression microRNA miR-200c was restored to the human TNBC mesenchymal-like BT549 cell line, which decreased 80% of the pan-EMT mesenchymal signature and restored 60% of the epithelial signature. In addition to decreasing proteins involved in immune-suppression via contact-dependent mechanisms, such as CD274 (PD-L1) and (CD273) PD-L2, Ingenuity Pathway and GSEA analysis revealed reduced expression of genes encoding enzymes, including Tryptophan 2,3-Dioxygenase (TDO2) and Heme Oxygenase-1 (HO-1), that generate immune-suppressive metabolites that function in a non-contact dependent manner. TNBC utilizes these and other immune-suppressive factors made by fetal trophoblasts to suppress the maternal immune system to achieve fetal tolerance during pregnancy, suggesting that TNBC may mimic a physiologically relevant mechanism to promote immune evasion. Hypothesis: TNBC metabolism is altered as part of an EMT program resulting in generation of immune-suppressive metabolites that promote metastasis by facilitating contact-independent immune evasion. Methods: Expression and activity of TDO2 and HO-1 were determined by UHPLC-MS for kynurenine (generated by TDO2) and bilirubin (generated by HO-1) in multiple TNBC cell lines. Single cell RNAseq was performed to identify genes co-expressed with these enzymes. To develop an immune competent preclinical model, doxycycline inducible miR-200c (TripZ-200c) was stably introduced into the Met-1 mammary carcinoma cell line derived from the MMTV-PyMT mouse model developed in FVB/NJ mice. Results were confirmed via transient miR-200c expression in 66cl-4 mammary carcinoma cells (from a spontaneous BALB/c tumor). Results: Restoration of miR-200c decreased TDO2 and HMOX1 (HO-1) by 80% and 40% respectively in Met-1 and 66cl-4 mammary carcinoma cells and reduced secretion of kynurenine and bilirubin. HO-1 was increased in lung metastasis from Met-1 and 66cl-4 cells when compared to cells prior to tail vein injection. ScRNAseq revealed specific gene expression signatures co-expressed with TDO2 and are being analyzed to better detect TDO2 in tumor tissues. In vivo studies are underway to investigate the effects of inhibiting tumor TDO2 and HO-1 on infiltrating lymphocyte composition, the anti-tumor immune response, and tumor progression. Conclusions: Restoration of miR-200c revealed EMT-associated alterations in tumor metabolism and consequent secretion of immune-suppressive metabolites produced by TDO2 and HO-1. We propose that targeting these contact-independent immune-suppressive factors may complement current contact-dependent therapeutic strategies (checkpoint inhibitors) to boost anti-tumor immune cell function and thereby prevent or decrease TNBC metastasis. Citation Format: Williams MM, Christenson JL, O'Neill K, Rogers TJ, Greene LI, Slansky J, Richer JK. Epithelial-mesenchymal transition promotes triple negative breast cancer immune suppression [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-04.

Pathobiology of the 129:Stat1\u2212/\u2212 mouse model of human age-related ER-positive breast cancer with an immune infiltrate-excluded phenotype

BackgroundStat1 gene-targeted knockout mice (129S6/SvEvTac-Stat1tm1Rds) develop estrogen receptor-positive (ER+), luminal-type mammary carcinomas at an advanced age. There is evidence for both host environment as well as tumor cell-intrinsic mechanisms to initiate tumorigenesis in this model. In this report, we summarize details of the systemic and mammary pathology at preneoplastic and tumor-bearing time points. In addition, we investigate tumor progression in the 129:Stat1−/− host compared with wild-type 129/SvEv, and we describe the immune cell reaction to the tumors.MethodsMice housed and treated according to National Institutes of Health guidelines and Institutional Animal Care and Use Committee-approved methods were evaluated by histopathology, and their tissues were subjected to immunohistochemistry with computer-assisted quantitative image analysis. Tumor cell culture and conditioned media from cell culture were used to perform macrophage (RAW264.7) cell migration assays, including the 129:Stat1−/−-derived SSM2 cells as well as control Met1 and NDL tumor cells and EpH4 normal cells.ResultsTumorigenesis in 129:Stat1−/− originates from a population of FoxA1+ large oval pale cells that initially appear and accumulate along the mammary ducts in segments or regions of the gland prior to giving rise to mammary intraepithelial neoplasias. Progression to invasive carcinoma is accompanied by a marked local stromal and immune cell response composed predominantly of T cells and macrophages. In conditioned media experiments, cells derived from 129:Stat1−/− tumors secrete both chemoattractant and chemoinhibitory factors, with greater attraction in the extracellular vesicular fraction and inhibition in the soluble fraction. The result appears to be recruitment of the immune reaction to the periphery of the tumor, with exclusion of immune cell infiltration into the tumor.Conclusions129:Stat1−/− is a unique model for studying the critical origins and risk reduction strategies in age-related ER+ breast cancer. In addition, it can be used in preclinical trials of hormonal and targeted therapies as well as immunotherapies.

Abstract 3610: Synergy between androgen receptor (AR)-targeting agents seviteronel (SEVI) or enzalutamide (ENZA) and mTOR or HER2 pathway-targeting agents in breast cancer

Abstract Background: AR is expressed in ~60% of HER2+ and up to 50% of triple negative breast cancer (TNBC) with growing evidence that AR plays a role in BC growth and survival. Next-generation AR-targeting agents including SEVI (selective CYP17-lyase and AR inhibitor) and ENZA (AR inhibitor) have shown promise in preclinical and clinical models of prostate cancer and are currently being evaluated in BC. Approved therapeutics targeting mTOR such as everolimus (EVEROL) and HER2-targeting agents provide significant clinical benefit in some BC patients; however, de novo and acquired resistance remain critical issues that may be partially attributed to compensatory AR action. Hypothesis: Due to cross-regulation between AR and the HER2/PI3K/mTOR pathway in BC, targeting AR in combination with approved agents for these pathways may improve responses. Methods: Human BC cell lines HER2+ (BT474, BT474-HR20, SKBR3) and TNBC (MDAMB453, BT549) and an AR+ TNBC mouse mammary carcinoma model (Met-1) were used. Cells were treated with multiple doses of AR-targeting agents SEVI or ENZA and EVEROL, alone or in combination and assayed over time using IncuCyte instrumentation and combination indices calculated using Calcusyn. Anchorage independence was tested by growth on soft agar. Pathway component genes and proteins were measured. In vivo, NOD/SCID mice with HER2+ trastuzumab-resistant BT474-HR20 xenografts were treated with ENZA, EVEROL, or the combination. Similar in vivo experiments with SEVI are ongoing. Results: Both SEVI and ENZA significantly inhibited proliferation of both HER2+ and TNBC cell lines. Both significantly inhibited growth on soft agar in l Met-1 cells (p=0.0005 for SEVI, p=0.032 for ENZA). Using 9 dose combinations of EVEROL with SEVI or ENZA, synergy was observed in multiple cell lines in vitro, but only in lines containing activating PIK3CA mutations. DHT induced a cell line-specific increase in pHER2 and/or pHER3 that was attenuated by AR inhibition. EVEROL alone caused an increase in total AR, pHER2, and pHER3, and these effects were also abrogated by AR inhibition. In vivo, ENZA inhibited BT474-HR20 xenografts, and combining ENZA with EVEROL decreased tumor viability more than either single agent (p<0.001, repeated measures ANOVA). Similar in vivo studies with SEVI are planned. Conclusion: Agents targeting AR synergize with EVEROL. Combination therapies targeting AR and the mTOR and HER2/HER3 pathways may provide benefit for HER2+ and TNBC patients. SEVI was a stronger inhibitor of anchorage-independent growth than ENZA and will thus be further investigated in combination with approved HER2/PI3K/mTOR pathway-directed therapeutics in vivo. This work was funded by DOD BCRP - Clinical Translational Award BC120183 W81XWH-13-1-0090 to JKR and 0091 to AE and R01 CA187733 to JKR Note: This abstract was not presented at the meeting. Citation Format: Michael A. Gordon, Nicholas D'Amato, Jessica Christenson, Beatrice Babbs, Haihua Gu, Julia Wulfkuhle, Emmanuel Petricoin, Anthoy Elias, Jennifer K. Richer. Synergy between androgen receptor (AR)-targeting agents seviteronel (SEVI) or enzalutamide (ENZA) and mTOR or HER2 pathway-targeting agents in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3610. doi:10.1158/1538-7445.AM2017-3610

Mint3 in bone marrow-derived cells promotes lung metastasis in breast cancer model mice

Breast cancer is one of the most common cancers in women in the world. Although breast cancer is well treatable at the early stage, patients with distant metastases show a poor prognosis. Data from recent studies using transplantation models indicate that Mint3/APBA3 might promote breast cancer malignancy. However, whether Mint3 indeed contributes to tumor development, progression, or metastasis in vivo remains unclear. To address this, here we examined whether Mint3 depletion affects tumor malignancy in MMTV-PyMT breast cancer model mice. In MMTV-PyMT mice, Mint3 depletion did not affect tumor onset and tumor growth, but attenuated lung metastases. Experimental lung metastasis of breast cancer Met-1 cells derived from MMTV-PyMT mice also decreased in Mint3-depleted mice, indicating that host Mint3 expression affected lung metastasis of MMTV-PyMT-derived breast cancer cells. Further bone marrow transplant experiments revealed that Mint3 in bone marrow-derived cells promoted lung metastasis in MMTV-PyMT mice. Thus, targeting Mint3 in bone marrow-derived cells might be a good strategy for preventing metastasis and improving the prognosis of breast cancer patients.

Erratum to: Non-metabolisable insulin glargine does not promote breast cancer growth in a mouse model of type 2 diabetes

Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25 U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.

MMTV-PyMT and Derived Met-1 Mouse Mammary Tumor Cells as Models for Studying the Role of the Androgen Receptor in Triple-Negative Breast Cancer Progression.

Triple-negative breast cancer (TNBC) has a faster rate of metastasis compared to other breast cancer subtypes, and no effective targeted therapies are currently FDA-approved. Recent data indicate that the androgen receptor (AR) promotes tumor survival and may serve as a potential therapeutic target in TNBC. Studies of AR in disease progression and the systemic effects of anti-androgens have been hindered by the lack of an AR-positive (AR+) immunocompetent preclinical model. In this study, we identified the transgenic MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mouse mammary gland carcinoma model of breast cancer and Met-1 cells derived from this model as tools to study the role of AR in breast cancer progression. AR protein expression was examined in late-stage primary tumors and lung metastases from MMTV-PyMT mice as well as in Met-1 cells by immunohistochemistry (IHC). Sensitivity of Met-1 cells to the AR agonist dihydrotestosterone (DHT) and anti-androgen therapy was examined using cell viability, migration/invasion, and anchorage-independent growth assays. Late-stage primary tumors and lung metastases from MMTV-PyMT mice and Met-1 cells expressed abundant nuclear AR protein, while negative for estrogen and progesterone receptors. Met-1 sensitivity to DHT and AR antagonists demonstrated a reliance on AR for survival, and AR antagonists inhibited invasion and anchorage-independent growth. These data suggest that the MMTV-PyMT model and Met-1 cells may serve as valuable tools for mechanistic studies of the role of AR in disease progression and how anti-androgens affect the tumor microenvironment.

Non-metabolisable insulin glargine does not promote breast cancer growth in a mouse model of type 2 diabetes.

Previous epidemiological studies have reported a potential link between insulin analogues and breast cancer; however, a prospective randomised controlled trial showed neutral effects of insulin glargine on cancer risk. Insulin glargine is metabolised in vivo to an M1 metabolite. A question remains whether a subset of individuals with slower rates of glargine metabolism or who are on high doses could, theoretically, have an increased risk of cancer progression if a tumour is already present. In this study, we aimed to determine whether a non-metabolisable form of insulin glargine induced murine breast cancer growth. A mouse model of type 2 diabetes (MKR) was used for these studies. MKR mice were injected with two murine mammary cancer cell lines: Mvt-1 cells (derived from MMTV-c-Myc/Vegf tumours) and Met1 cells (derived from MMTV-polyoma virus middle T antigen tumours). Mice were treated with 25U/kg per day of the long-acting insulin analogues, insulin glargine, insulin detemir, insulin degludec or non-metabolisable glargine, or vehicle. No difference in tumour growth was seen in terms of tumour size after insulin glargine, detemir, degludec or vehicle injections. Non-metabolisable glargine did not increase tumour growth compared with insulin glargine or vehicle. Insulin glargine and non-metabolisable glargine led to insulin receptor phosphorylation in vivo rather than IGF-1 receptor phosphorylation. These results demonstrate that in a mouse model of type 2 diabetes, at high concentrations, basal insulin analogues and a non-metabolisable glargine analogue do not promote the progression of breast tumours.

Epithelial‐mesenchymal transition during invasion of cutaneous squamous cell carcinoma is paralleled by AKT activation

Epithelial-mesenchymal transition (EMT) is required for tumour invasion and dissemination to occur. To investigate EMT during invasion of cutaneous squamous cell carcinoma (cSCC) and the involvement of AKT. Using a tissue microarray, we measured expression of EMT-markers and AKT activation in 140 samples from patients with skin cancer and matched samples of normal skin adjacent to cSCC in cSCC in situ (cSCCIS) and in invasive cSCC. We investigated EMT using functional assays and the expression of EMT markers in an isogenic skin cancer progression model using cell lines derived from dysplastic forehead skin (PM1), primary invasive cSCC (MET1) and its lymph node metastasis (MET4). This model was used to investigate AKT-specific inhibition of the EMT process. In comparison with normal skin, and normal skin plus cSCCIS, the invasive cSCCs show significantly increased vimentin expression, decreased E-cadherin expression and increased expression of the active form of AKT. In the cell culture model, the primary MET1 cells display the lowest adhesion potential, the highest migratory and invasive ability through a Matrigel-coated porous membrane, the highest expression of EMT markers vimentin and Slug and the lowest expression of the epithelial marker E-cadherin. Pharmacological AKT inhibition in this model suppressed EMT mechanisms. AKT may serve as a therapeutic target to avoid dissemination of cSCC cells.

Success of measles virotherapy in ATL depends on type I interferon secretion and responsiveness

Adult T cell leukemia/lymphoma (ATL) is a highly aggressive CD4+/CD25+ T-cell malignancy caused by human T cell lymphotropic virus type 1 (HTLV-1). Previous studies in the MET-1 cell/NOD/SCID mouse model of ATL demonstrated that MET-1 cells are very susceptible to measles virus (MV) oncolytic therapy. To further evaluate the potential of MV therapy in ATL, the susceptibility of several HTLV-1 transformed CD4+ T cell lines (MT-1, MT-2, MT-4 and C8166-45) as well as HTLV-1 negative CD4+ T cell lines (Jurkat and CCRF-CEM) to infection with MV was tested in vitro. All cell lines were permissive to MV infection and subsequent cell death, except MT-1 and CCRF-CEM cells which were susceptible and permissive to MV infection, but resistant to cell death. The resistance to MV-mediated cell death was associated with IFNβ produced by MT-1 and CCRF-CEM cells. Inhibition of IFNβ rendered MT-1 and CCRF-CEM cells susceptible to MV-mediated cell death. Cells susceptible to MV-induced cell death did not produce nor were responsive to IFNβ. Upon infection with Newcastle Disease Virus (NDV), MT-1 and CCRF-CEM but not the susceptible cell lines up-regulated pSTAT-2. In vivo, treatment of tumors induced by MT-1 cell lines which produce IFNβ demonstrated only small increases in mean survival time, while only two treatments prolonged mean survival time in mice with MET-1 tumors deficient in type I interferon production. These results indicate that type I interferon production is closely linked with the inability of tumor cells to respond to type I interferon. Screening of tumor cells for type I interferon could be a useful strategy to select candidate patients for MV virotherapy.

Tumor Microenvironment Regulates Metastasis and Metastasis Genes of Mouse MMTV-PymT Mammary Cancer Cells In Vivo

Metastasis is the primary cause of death in breast cancer patients, yet there are challenges to modeling this process in vivo. The goal of this study was to analyze the effects of injection site on tumor growth and metastasis and gene expression of breast cancer cells in vivo using the MMTV-PymT breast cancer model (Met-1 cells). Met-1 cells were injected into 5 sites (subcutaneous, mammary fat pad, tail vein, intracardiac, and intratibial), and tumors and metastases were monitored using bioluminescent imaging and confirmed with gross necropsy and histopathology. Met-1 tumors were analyzed based on morphology and changes in gene expression in each tissue microenvironment. There were 6 permissible sites of Met-1 tumor growth (mammary gland, subcutis, lung, adrenal gland, ovary, bone). Met-1 cells grew faster in the subcutis compared to mammary fat pad tumors (highest Ki-67 index). Morphologic differences were evident in each tumor microenvironment. Finally, 7 genes were differentially expressed in the Met-1 tumors in the 6 sites of growth or metastasis. This investigation demonstrates that breast cancer progression and metastasis are regulated by not only the tumor cells but also the experimental model and unique molecular signals from the tumor microenvironment.

Abstract 1729: Mitochondrial necrosis pathway drives tumor progression.

Abstract Necrosis is a prominent feature of aggressive and invasive cancers. Previous clinical/epidemiological studies have suggested an association between the extent of tumor necrosis and poor prognosis and survivorship for some cancers. Evasion of apoptosis is widely accepted as a hallmark of cancer. However, whether necrotic cell death plays no role, limits, or drives carcinogenesis has not been studied in any cancer. Recent work suggests that necrosis may be an actively regulated form of cell death. In contrast to apoptosis where permeabilization of the outer mitochondrial membrane is the defining event, the central event in necrosis is the opening of a pore in the inner mitochondrial membrane termed the mitochondrial permeability transition pore (mPTP). Cyclophilin D, a peptidyl prolyl cis-trans isomerase in the mitochondrial matrix, encoded by the gene ppif, is critical for opening of the mPTP. Stable expression of ppif shRNA was used to deplete cyclophilin D in Met-1 cells, a metastatic breast cancer cell line. We first confirmed that knockdown of ppif in Met-1 cells abrogated necrosis, but not apoptosis. When these cells were transplanted into the mammary glands of wildtype FVB mice, tumors resulting from cells that express ppif shRNA were markedly smaller, with decreased volume and mass, than those derived from cells expressing control shRNA. Reconstitution of ppif knockdown cells with wild type cyclophilin D, but not an enzymatically inactive mutant, rescued tumor volume and mass. As expected, tumors arising from ppif knockdown cells exhibited less necrosis, while rates of apoptosis were unchanged. Moreover, rates of BrdU incorporation were significantly decreased in these tumors. Necrotic cell death is characterized by loss of plasma membrane integrity resulting in a marked inflammatory response. Neutrophils and macrophages recruited to tumors often secrete growth/survival/angiogenic factors that promote tumor growth and metastasis. Consistent with this model, both macrophage and neutrophil recruitment were reduced in tumors derived from ppif knockdown cells. In addition, rates of spontaneous pulmonary metastasis were markedly decreased in mice harboring tumors derived from ppif knockdown cells compared with controls. Our data suggest that, in contrast to apoptotic cell death which limits carcinogenesis, necrotic cell death actually drives disease progression by altering the tumor microenvironment. These findings may have therapeutic implications in that they suggest that inhibition - rather than promotion - of necrotic cell death may provide a novel treatment paradigm for some cancers. Citation Format: Melissa K. James, Richard N. Kitsis. Mitochondrial necrosis pathway drives tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1729. doi:10.1158/1538-7445.AM2013-1729

IGF1 dependence of dietary energy balance effects on murine Met1 mammary tumor progression, epithelial-to-mesenchymal transition, and chemokine expression

Luminal breast tumors with little or no estrogen receptor α expression confer poor prognosis. Using the Met1 murine model of luminal breast cancer, we characterized the IGF1-dependency of diet-induced obesity (DIO) and calorie restriction (CR) effects on tumor growth, growth factor signaling, epithelial-to-mesenchymal transition (EMT), and chemokine expression. Liver-specific IGF1-deficient (LID) and littermate control (LC) mice were administered control, DIO, or 30% CR diets for 3 months before orthotopic injection of Met1 cells. Tumors grew for 1 month and then were assessed for Akt pathway activation and mRNA expression of chemokine and EMT constituents. LID mice, regardless of diet, displayed reduced Met1 tumor growth and downregulated Akt, EMT, and chemokine pathways. CR, relative to control, reduced serum IGF1 and Met1 tumor growth in LC (but not LID) mice. DIO, relative to control, increased Met1 tumor growth and chemokine expression in LID mice, and had no effect on serum IGF1 or pAkt or cyclin D1 expression in either genotype. Thus, circulating IGF1 (in association with Akt, EMT, and chemokines) regulated Met1 tumor growth. While the anticancer effects of CR were largely IGF1-dependent, the procancer effects of DIO manifested only when circulating IGF1 levels were low. Thus, in a murine model of luminal breast cancer, IGF1 and its downstream signaling pathway, EMT, and chemokines present possible mechanistic regulatory targets. Transplanted MMTV1 Wnt1 mammary tumor growth was also reduced in LID mice, relative to LC mice, suggesting that the IGF1 effects on mammary tumor growth are not limited to Met1 tumors.

Pattern of sensitivity of progressive cutaneous squamous cell carcinoma cells to UVB and oxidative stress-induced cell death

Previous investigations have demonstrated that isogenic cutaneous squamous cell carcinoma cell lines (cSCC), isolated from highly dysplastic skin (PM1), primary invasive SCC (MET1) and its lymph node metastasis (MET4), show an increasing resistance to cisplatin-induced apoptosis in the increasingly malignant MET1 and MET4 cells. To investigate whether cell death sensitivity in progressive stages of skin carcinogenesis is dependent on the kind of stress we examined the sensitivity of PM1, MET1 and MET4 cells to apoptosis in response to a single UVB-dose (mixture of genotoxic and oxidative stress), or to hydrogen peroxide and hypericin photodynamic treatment (both pure oxidative stresses). MET1 cells, followed by the MET4 cells, were more sensitive to UVB, resulting in more cell death and more apoptosis in comparison with the PM1 cells. A similar pattern of sensitivity was observed when we exposed the SCC cells to hydrogen peroxide or hypericin photodynamic treatment, which both generate mainly oxidative stress. The MET1 cells were the most sensitive to all stresses examined. The pattern of cell death sensitivity in a model of progressive cutaneous squamous cell carcinoma is dependent on the kind of stress. While more advanced skin cancer cells like MET1 and MET4 cells lose their sensitivity to the chemotherapeutic agent cisplatin, they remain sensitive to hydrogen peroxide or physical treatments, which induce major oxidative stress. This differential sensitivity could have implications for the treatment of advanced cSCC.