Non-malignant Epithelial Cells Research Articles

Hypoxia-driven metabolic reprogramming of adipocytes fuels cancer cell proliferation.

Obesity increases the risk of certain cancers, especially tumours that reside close to adipose tissue (breast and ovarian metastasis in the omentum). The obesogenic and tumour micro-environment share a common pathogenic feature, oxygen deprivation (hypoxia). Here we test how hypoxia changes the metabolome of adipocytes to assist cancer cell growth. Human and mouse breast and ovarian cancer cell lines were co-cultured with human and mouse adipocytes respectively under normoxia or hypoxia. Proliferation and lipid uptake in cancer cells were measured by commercial assays. Metabolite changes under normoxia or hypoxia were measured in the media of human adipocytes by targeted LC/MS. Hypoxic cancer-conditioned media increased lipolysis in both human and mouse adipocytes. This led to increased transfer of lipids to cancer cells and consequent increased proliferation under hypoxia. These effects were dependent on HIF1α expression in adipocytes, as mouse adipocytes lacking HIF1α showed blunted responses under hypoxic conditions. Targeted metabolomics of the human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes media revealed that culture with hypoxic-conditioned media from non-malignant mammary epithelial cells (MCF10A) can alter the adipocyte metabolome and drive proliferation of the non-malignant cells. Here, we show that hypoxia in the adipose-tumour microenvironment is the driving force of the lipid uptake in both mammary and ovarian cancer cells. Hypoxia can modify the adipocyte metabolome towards accelerated lipolysis, glucose deprivation and reduced ketosis. These metabolic shifts in adipocytes could assist both mammary epithelial and cancer cells to bypass the inhibitory effects of hypoxia on proliferation and thrive.

SLC35A2 Deficiency Promotes an Epithelial-to-Mesenchymal Transition-like Phenotype in Madin-Darby Canine Kidney Cells.

In mammalian cells, SLC35A2 delivers UDP–galactose for galactosylation reactions that take place predominantly in the Golgi lumen. Mutations in the corresponding gene cause a subtype of a congenital disorder of glycosylation (SLC35A2-CDG). Although more and more patients are diagnosed with SLC35A2-CDG, the link between defective galactosylation and disease symptoms is not fully understood. According to a number of reports, impaired glycosylation may trigger the process of epithelial-to-mesenchymal transition (EMT). We therefore examined whether the loss of SLC35A2 activity would promote EMT in a non-malignant epithelial cell line. For this purpose, we knocked out the SLC35A2 gene in Madin–Darby canine kidney (MDCK) cells. The resulting clones adopted an elongated, spindle-shaped morphology and showed impaired cell–cell adhesion. Using qPCR and western blotting, we revealed down-regulation of E-cadherin in the knockouts, while the fibronectin and vimentin levels were elevated. Moreover, the knockout cells displayed reorganization of vimentin intermediate filaments and altered subcellular distribution of a vimentin-binding protein, formiminotransferase cyclodeaminase (FTCD). Furthermore, depletion of SLC35A2 triggered Golgi compaction. Finally, the SLC35A2 knockouts displayed increased motility and invasiveness. In conclusion, SLC35A2-deficient MDCK cells showed several hallmarks of EMT. Our findings point to a novel role for SLC35A2 as a gatekeeper of the epithelial phenotype.

Circulating microRNAs as molecular biomarkers for lung adenocarcinoma.

The potential of microRNAs (miRNAs) as molecular tumor biomarkers for early diagnosis and prognosis in lung cancer is still unclear. To analyze expression of miRNAs in A549 lung adenocarcinoma (LUAD) cells and in primary, non-malignant bronchial epithelial (BE) cells from healthy donors. To analyze the most prominently deregulated miRNAs in plasma samples of LUAD patients and healthy donors. The expression of 752 miRNAs in LUAD and BE cells was assessed by RT-qPCR with mean-centering restricted normalization. The relative plasma levels of 18 miRNAs in LUAD patients and healthy donors were analyzed using RT-qPCR and normalized to miR-191-5p and miR-16-3p. Putative interactions between miRNAs and their target genes were investigated in silico. Out of 752 miRNAs, 37 miRNAs were significantly deregulated in A549 cells compared to BE cells. MiR-15b-3p, miR-148a-3p, miR-193b-3p, and miR-195-5p were significantly deregulated in plasma samples of LUAD patients compared to donors. The target genes of those four miRNAs are involved in essential mechanisms in cancer development and progression. There are substantial differences between cancer and control miRNA expression in vitro and in plasma samples of LUAD patients compared to healthy donors. Four deregulated miRNAs are promising as a diagnostic biomarker for adenocarcinoma of the lung.

Field carcinogenesis and biological significance of the potential of the bystander effect: carcinogenesis, therapeutic response, and tissue regeneration.

The "bystander effect" is a transmission phenomenon mediating communication from target to non-target cells, as well as cell-to-cell interactions between neighboring and distantly located cells. In this narrative review, we describe the fundamental and clinical significance of the bystander effect with respect to cell-to-cell interactions in carcinogenesis, therapeutic response, and tissue regeneration. In carcinogenesis, the bystander effect mediates communications between tumor microenvironments and non-malignant epithelial cells and has been suggested to impact heterogeneous tumorigenic cells in tumors and cancerized fields. In therapeutic response, the bystander effect mediates communications between drug-sensitive and drug-resistant cells and may transmit both drug efficacy and resistance. Therefore, control of therapeutic response transmission via the bystander effect might offer a promising future cancer treatment. Finally, in tissue regeneration, circulating cells and stromal cells may differentiate into various cells for the purpose of tissue regeneration under direction of the bystander effect arising from surrounding cells in a defective space. We hope that the findings we present will promote the development of innovative cancer therapies and tissue regeneration methodologies from the viewpoint of cell-to-cell interactions through the bystander effect.

How does direct current atmospheric pressure glow discharge application influence on physicochemical, nutritional, microbiological, and cytotoxic properties of orange juice?

We proposed an innovative and economic method for rapid production of functionalized orange juice (OJ) with excellent nutritional properties, prolonged shelf life, and safe consumption. To reach this goal, we have employed direct current atmospheric pressure glow discharge, generated in contact with a flowing liquid cathode (FLC-dc-APGD) in a highly-throughput reaction-discharge system. It was found that controlled FLC-dc-APGD-treatment of OJ lead to increase the concentration of selected metals and phenolic compounds.The so-obtained OJ had the same qualitative composition of fragrance as the untreated one, however, its shelf life was prolonged up to 26 days. Furthermore, OJ exposed to FLC-dc-APGD-treatment did not exhibit any cytotoxic properties towards non-malignant human intestinal epithelial cell lines. On the other hand, the induction of cell cytotoxicity was observed in human colorectal adenocarcinoma cells line after FLC-dc-APGD-treated OJ application. We truly believe that produced by us functionalized OJ might be a tempting alternative to classic, non-treated by FLC-dc-APGD OJ.

Early lung carcinogenesis and tumor microenvironment observed by single-cell transcriptome analysis

With the increasing interest in health screening with chest CT Ground-glass nodule (GGN) has become one of the common lung lesions encountered in daily medical practice. Because lung adenocarcinoma in the form of GGN is an ideal model for studying early lung carcinogenesis, 11 GGN and normal lung specimens from 6 never smoker patients were studied by single-cell RNA sequencing. Lung cancer cells showed enrichment of gene sets related to small vesicle processing and surfactant homeostasis compared to non-malignant lung epithelial cells, suggesting the dysregulation of surfactant pathway may be involved in early lung carcinogenesis. Along with cancer-associated fibroblasts showing enrichment of gene sets involved in negative regulation of protein kinase activity and negative regulation of endothelial cell proliferation, tumor microenvironment (TME) was dominated by infiltration of TNFRSF4+/TNFRSF18+/CTLA4+ regulatory T cells (Treg) and depletion of CD8+ cytotoxic T cells (TC) and γδTC. Majority of mucosa-associated lymphoid tissue B cells (BCs) and follicular BCs were detected within tumor tissue, which was associated with CXCL13 overexpressed in intratumoral Tregs and CD4+ memory TCs. Coordination of components of the TME towards immune evasion is governed by Tregs from the onset of lung cancer, requiring unremitting efforts to target and overcome them. This provision of information on changes in cancer cell-specific biomarkers and TME using early lung cancer from never smokers will provide new insight into early lung carcinogenesis and useful targets for treatment.

The non-canonical mechanism of ER stress-mediated progression of prostate cancer

BackgroundThe development of persistent endoplasmic reticulum (ER) stress is one of the cornerstones of prostate carcinogenesis; however, the mechanism is missing. Also, alcohol is a physiological ER stress inducer, and the link between alcoholism and progression of prostate cancer (PCa) is well documented but not well characterized. According to the canonical model, the mediator of ER stress, ATF6, is cleaved sequentially in the Golgi by S1P and S2P proteases; thereafter, the genes responsible for unfolded protein response (UPR) undergo transactivation.MethodsCell lines used were non-malignant prostate epithelial RWPE-1 cells, androgen-responsive LNCaP, and 22RV1 cells, as well as androgen-refractory PC-3 cells. We also utilized PCa tissue sections from patients with different Gleason scores and alcohol consumption backgrounds. Several sophisticated approaches were employed, including Structured illumination superresolution microscopy, Proximity ligation assay, Atomic force microscopy, and Nuclear magnetic resonance spectroscopy.ResultsHerein, we identified the trans-Golgi matrix dimeric protein GCC185 as a Golgi retention partner for both S1P and S2P, and in cells lacking GCC185, these enzymes lose intra-Golgi situation. Progression of prostate cancer (PCa) is associated with overproduction of S1P and S2P but monomerization of GCC185 and its downregulation. Utilizing different ER stress models, including ethanol administration, we found that PCa cells employ an elegant mechanism that auto-activates ER stress by fragmentation of Golgi, translocation of S1P and S2P from Golgi to ER, followed by intra-ER cleavage of ATF6, accelerated UPR, and cell proliferation. The segregation of S1P and S2P from Golgi and activation of ATF6 are positively correlated with androgen receptor signaling, different disease stages, and alcohol consumption. Finally, depletion of ATF6 significantly retarded the growth of xenograft prostate tumors and blocks production of pro-metastatic metabolites.ConclusionsWe found that progression of PCa associates with translocation of S1P and S2P proteases to the ER and subsequent ATF6 cleavage. This obviates the need for ATF6 transport to the Golgi and enhances UPR and cell proliferation. Thus, we provide the novel mechanistic model of ATF6 activation and ER stress implication in the progression of PCa, suggesting ATF6 is a novel promising target for prostate cancer therapy.

Ursolic Acid Inhibits Breast Cancer Metastasis by Suppressing Glycolytic Metabolism via Activating SP1/Caveolin-1 Signaling.

Breast cancer remains the most common malignancy and the leading causality of cancer-associated mortality among women worldwide. With proven efficacy, Oldenlandia diffusa has been extensively applied in breast cancer treatment in Traditional Chinese Medicine (TCM) for thousands of years. However, the bioactive compounds of Oldenlandia diffusa accounting for its anti-breast cancer activity and the underlying biological mechanisms remain to be uncovered. Herein, bioactivity-guided fractionation suggested ursolic acid as the strongest anti-breast cancer compound in Oldenlandia diffusa. Ursolic acid treatment dramatically suppressed the proliferation and promoted mitochondrial-mediated apoptosis in breast cancer cells while brought little cytotoxicities in nonmalignant mammary epithelial cells in vitro. Meanwhile, ursolic acid dramatically impaired both the glycolytic metabolism and mitochondrial respiration function of breast cancer cells. Further investigations demonstrated that ursolic acid may impair the glycolytic metabolism of breast cancer cells by activating Caveolin-1 (Cav-1) signaling, as Cav-1 knockdown could partially abrogate the suppressive effect of ursolic acid on that. Mechanistically, ursolic acid could activate SP1-mediated CAV1 transcription by promoting SP1 expression as well as its binding with CAV1 promoter region. More meaningfully, ursolic acid administration could dramatically suppress the growth and metastasis of breast cancer in both the zebrafish and mouse xenotransplantation models of breast cancer in vivo without any detectable hepatotoxicity, nephrotoxicity or hematotoxicity. This study not only provides preclinical evidence supporting the application of ursolic acid as a promising candidate drug for breast cancer treatment but also sheds novel light on Cav-1 as a druggable target for glycolytic modulation of breast cancer.

Hydralazine and Panobinostat Attenuate Malignant Properties of Prostate Cancer Cell Lines.

Among the well-established alterations contributing to prostate cancer (PCa) pathogenesis, epigenetics is an important player in its development and aggressive disease state. Moreover, since no curative therapies are available for advanced stage disease, there is an urgent need for novel therapeutic strategies targeting this subset of patients. Thus, we aimed to evaluate the combined antineoplastic effects of DNA methylation inhibitor hydralazine and histone deacetylase inhibitors panobinostat and valproic acid in several prostate cell lines. The effect of these drugs was assessed in four PCa (LNCaP, 22Rv1, DU145 and PC-3) cell lines, as well as in non-malignant epithelial (RWPE-1) and stromal (WPMY-1) cell lines, using several assays to evaluate cell viability, apoptosis, proliferation, DNA damage and clonogenic potential. We found that exposure to each epidrug separately reduced viability of all PCa cells in a dose-dependent manner and that combined treatments led to synergic growth inhibitory effects, impacting also on colony formation, invasion, apoptotic and proliferation rates. Interestingly, antitumoral effects of combined treatment were particularly expressive in DU145 cells. We concluded that hydralazine and panobinostat attenuate malignant properties of PCa cells, constituting a potential therapeutic tool to counteract PCa progression.

Abstract 92: Clonal evolution of proliferative lesions into breast cancers

Abstract Introduction: Proliferative lesions in the breast have been implicated in the development of invasive breast cancer. Previous studies showed that adjacent atypical proliferative lesions and breast cancers shared common genetic alterations, suggesting that these evolved from the same ancestral cell. However, the clonal structure of atypical proliferative lesions and their clonal dynamics during progression to cancer are poorly understood. In this study, we compared genetic profiles of proliferative lesions and cancers from the same patients to illustrate the clonal evolution of cancer from a non-malignant epithelial cell. Methods: Multiple samples were collected from various lesions with different disease-level located in the cancer-borne breast, using micro-dissection from formalin-fixed, paraffin-embedded surgical specimens. Somatic mutations and copy number alterations (CNAs) were evaluated by whole exome sequencing. Results: A total of 39 samples from 7 premenopausal females carrying estrogen receptor-positive cancers were analyzed, where the samples were obtained from proliferative lesions (without/with atypia) (N = 3 and 10, respectively), and non-invasive (N = 22) and invasive (N = 4) cancers. The number of somatic mutations per sample increased with disease progression (ranging from 7 to 311). Two cases with bilateral cancers had a pathogenic germline mutation of either BRCA2 or TP53, where no somatic mutations or CNAs were shared by individual proliferative lesions, suggesting multifocal independent cancerous evolutions. By contrast, in the remaining five unilateral cases, no pathogenic germline mutations were detected, but all proliferative lesions, which were separated by a distance of 7 - 70 mm, shared one or more driver alterations, such as an AKT1 mutation, a GATA3 mutation, a CBFB mutation, a PTEN mutation and concurrent 1q gain and 16q loss, while harboring private mutations/CNAs of their own. The analysis of phylogenic trees based on the number of shared mutations predicted an early origin of these founder mutations, which frequently predated decades before the onset of cancer. Conclusions: Our results suggest that early breast cancer development is shaped by the evolution of multiple precancerous clones. In hereditary cases, this process is thought to be substantially promoted multi-focally within the entire breasts, due to innate genomic instability in each mammary epithelial cell for pathogenic germline mutations. By contrast, in sporadic cases, the ancestral cell which has acquired a founder genetic alteration expands macroscopically long before the onset of cancer. These expanded clones work as the niche predisposed to additional mutations, in which branching evolution occurs multi-focally and raises up multiple proliferative lesions, from which invasive cancer finally evolves. Our findings provide unique insight into the early development of breast cancer. Citation Format: Tomomi Nishimura, Nobuyuki Kakiuchi, Kenichi Yoshida, Yasuhide Takeuchi, Hirona Maeda, Yusuke Shiozawa, Masahiro M. Nakagawa, Yotaro Ochi, Yukiko Kawata, Kosuke Aoki, Masahiro Hirata, Tatsuki R. Kataoka, Takaki Sakurai, Satoko Baba, Yuichi Shiraishi, Kenichi Chiba, Kengo Takeuchi, Hironori Haga, Satoru Miyano, Masakazu Toi, Seishi Ogawa. Clonal evolution of proliferative lesions into breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 92.

Influence of Terminal Functionality on the Crystal Packing Behaviour and Cytotoxicity of Aromatic Oligoamides.

The synthesis and characterization of three aromatic oligoamides, constructed from the same pyridyl carboxamide core but incorporating distinct end groups of acetyl (Ac) 1, tert-butyloxycarbonyl (Boc) 2 and amine 3 is reported. Single crystal X-ray diffraction analysis of 1–3 and a dimethylsulfoxide (DMSO) solvate of 2 (2-DMSO), has identified the presence of a range of intra- and intermolecular interactions including N-H⋯N, N-H⋯O=C and N-H⋯O=S(CH3)2 hydrogen-bonding interactions, C-H⋯π interactions and off-set, face-to-face stacking π-π interactions that support the variety of slipped stack, herringbone and cofacial crystal packing arrangements observed in 1–3. Additionally, the cytotoxicity of this series of aromatic oligoamides was assessed against two human ovarian (A2780 and A2780cisR), two human breast (MCF-7 and MDA-MB-231) cancerous cell lines and one non-malignant human epithelial cell line (PNT-2), to investigate the influence of the terminal functionality of these aromatic oligoamides on their biological activity. The chemosensitivity results highlight that modification of the terminal group from Ac to Boc in 1 and 2 leads to a 3-fold increase in antiproliferative activity against the cisplatin-sensitive ovarian carcinoma cell line, A2780. The presence of the amine termini in 3 gave the only member of the series to display activity against the cisplatin-resistance ovarian carcinoma cell line, A2780cisR. Compound 2 is the lead candidate of this series, displaying high selectivity towards A2780 cancer cells when compared to non-malignant PNT-2 cells, with a selectivity index value >4.2. Importantly, this compound is more selective towards A2780 (cf. PNT-2) than the clinical platinum drugs oxaliplatin by > 2.6-fold and carboplatin by > 1.6-fold.

Rac1-mediated sustained β4 integrin level develops reattachment ability of breast cancer cells after anchorage loss.

Previously, we reported that non‐apoptotic cell death was induced in non‐malignant mammary epithelial cells (HMECs) upon loss of anchorage during 48 h incubation in suspension. In this study, we examined HMECs in suspension at an earlier time point and found that most of them lost attachment ability to substrata when replated, although >80% were alive. This suggested that HMECs lost reattachment ability (RA) prior to cell death upon detachment. Concomitant with the loss of RA, a decrease in the levels of β1 and β4 integrin was observed. In sharp contrast, breast cancer cells retained integrin levels, reattached to substrata, and formed colonies after exposure to anchorage loss as efficiently as those maintained under adherent conditions. Such RA of cancer cells is essential for the metastatic process, especially for establishing adhesion contact with ECM in the secondary organ after systemic circulation. Further analysis suggested that sustained levels of β4 integrin, which was mediated by Rac1, was critical for RA after anchorage loss and lung metastasis of breast cancer cells. In the cancer cells, persistent Rac1 activity enhanced escape of β4 integrin from lysosomal degradation depending on actin‐related protein 2/3 and TBC1D2, a GTPase‐activating protein of Rab7 GTPase. Notably, simultaneous high expression of ITGB4 and RAC1 was associated with poor prognosis in patients with breast cancer. Therefore, β4 integrin and Rac1 are attractive therapeutic targets to eliminate RA in cancer cells, thereby preventing the initial step of colonization at the secondary organ during metastasis.

Dynamic patterns of DNA methylation in the normal prostate epithelial differentiation program are targets of aberrant methylation in prostate cancer

Understanding the epigenetic control of normal differentiation programs might yield principal information about critical regulatory states that are disturbed in cancer. We utilized the established non-malignant HPr1-AR prostate epithelial cell model that upon androgen exposure commits to a luminal cell differentiation trajectory from that of a basal-like state. We profile the dynamic transcriptome associated with this transition at multiple time points (0 h, 1 h, 24 h, 96 h), and confirm that expression patterns are strongly indicative of a progressive basal to luminal cell differentiation program based on human expression signatures. Furthermore, we establish dynamic patterns of DNA methylation associated with this program by use of whole genome bisulfite sequencing (WGBS). Expression patterns associated with androgen induced luminal cell differentiation were found to have significantly elevated DNA methylation dynamics. Shifts in methylation profiles were strongly associated with Polycomb repressed regions and to promoters associated with bivalency, and strongly enriched for binding motifs of AR and MYC. Importantly, we found that dynamic DNA methylation patterns observed in the normal luminal cell differentiation program were significant targets of aberrant methylation in prostate cancer. These findings suggest that the normal dynamics of DNA methylation in luminal differentiation contribute to the aberrant methylation patterns in prostate cancer.

Expanded human NK cells armed with CAR uncouple potent anti-tumor activity from off-tumor toxicity against solid tumors

SummaryDespite the remarkable success of chimeric antigen receptor (CAR)-T cells against hematologic malignancies, severe off-tumor effects have constrained their use against solid tumors. Recently, CAR-engineered natural killer (NK) cells have emerged as an effective and safe alternative. Here, we demonstrate that HER2 CAR-expression in NK cells from healthy donors and patients with breast cancer potently enhances their anti-tumor functions against various HER2-expressing cancer cells, regardless of MHC class I expression. Moreover, HER2 CAR-NK cells exert higher cytotoxicity than donor-matched HER2 CAR-T cells against tumor targets. Importantly, unlike CAR-T cells, HER2 CAR-NK cells do not elicit enhanced cytotoxicity or inflammatory cytokine production against non-malignant human lung epithelial cells with basal HER2 expression. Further, HER2 CAR-NK cells maintain high cytotoxic function in the presence of immunosuppressive factors enriched in solid tumors. These results show that CAR-NK cells may be a highly potent and safe source of immunotherapy in the context of solid tumors.

PH- and miRNA-Responsive DNA-Tetrahedra/Metal-Organic Framework Conjugates: Functional Sense-and-Treat Carriers.

The synthesis of stimuli-responsive hybrid structures composed of drug-loaded UiO-66 metal-organic framework nanoparticles, NMOFs, locked by DNA tetrahedra gates is presented. The hybrid systems combine the high loading capacity of drugs in the porous NMOFs and the effective cell permeation properties of the DNA tetrahedra. The nucleic acid-functionalized UiO-66 NMOFs are loaded with drugs (doxorubicin, DOX, or camptothecin, CPT) or with dyes as drug models (Rhodamine 6G or fluorescein) and used to prepare stimuli-responsive carriers. In this study, two different stimuli-responsive NMOFs are presented. One system introduces the drug-loaded NMOFs locked by pH-responsive DNA tetrahedra. At acidic pH values, the gating tetrahedra are dissociated from the NMOFs through the formation of i-motif structures, resulting in the unlocking of the NMOFs and the release of the drugs. In addition, the tetrahedra gates are modified with AS1411 aptamer tethers, and these target the drug-loaded NMOFs to nucleolin receptors overexpressed in certain malignant cells. A second system involves the preparation of NMOFs loaded with drugs/dyes and gated by the microRNA (miRNA)-responsive tetrahedra (miRNA-21 or miRNA-155). In the presence of miRNAs, the dissociation of miRNA-responsive tetrahedra from the NMOFs leads to the unlocking of the NMOFs and the release of the loads. Further developments of the miRNA-responsive tetrahedra-gated hybrid carriers include the following. (i) By appropriate engineering of the miRNA gating units, the exonuclease III (Exo III)-amplified unlocking of the carriers, through the regeneration of the miRNA triggers, and the enhanced release of the loaded drugs are demonstrated. (ii) By applying mixtures of miRNA-21-responsive DNA tetrahedra-gated DOX-loaded NMOFs and miRNA-155-responsive DNA tetrahedra-gated CPT-loaded NMOFs, the multiplexed miRNA-21/miRNA-155-dictated release of the drugs is demonstrated. As compared to the analog DNA duplex-modified NMOFs, DNA tetrahedra-gated, drug-loaded NMOFs permeation into malignant MDA-MB-231 breast cancer cells presents more effective cell permeation. Effective and selective cytotoxicity toward the malignant cells, as compared to nonmalignant epithelial MCF-10A breast cells, is demonstrated due to the acidic pH, present in cancer cells, or the miRNA-21, present in MDA-MB-231 malignant cells.

Abstract IA003: Radiotherapy effects on tumor microenvironment

Abstract Radiation therapy (RT) exerts multiple effects on the tumor and its microenvironment, affecting immunity, metabolism and the microbiome (Heather M. McGee et al. Clin Cancer Res 2019;25:2969-2974). Radiation has profound and sustained effects on non-malignant tissue, including epithelial to mesenchymal transition (EMT) and carcinogenesis. Seminal experiments from Barcellos-Hoff' s lab originally demonstrated the capacity of RT to induce EMT of non-malignant epithelial cells (Andarawewa, KL et al: Cancer Res 2007; 67: (18). September 15, 2007). Burma et al demonstrated in an in vivo model of gliomas engineered after loss of one allele of Trp53 and Pten, that mice were highly susceptible to radiation-induced gliomagenesis in a linear energy transfer (LET)-dependent manner, possibly dependent on the increasing degree of irreparable DNA breaks with increasing LET (Todorova PK et al Cancer Res 2007; 67: (18). September 15, 2007). Interestingly, the histopathology of RT-induced high grade gliomas in this model mimics that of human glioblastoma, with similar patterns of necrosis, pseudo-palisading and nuclear atypia. This effect has translational implications, in view of the fact that the adjacent irradiated brain after standard post-operative RT to GBM is the most common site of recurrence (Gupta K and Burns TC: Frontiers in Oncology, 2018). Radiation is a powerful inducer of immunogenic cell death (ICD) of cancer cells, by releasing of DAMPs such as high mobility group box protein 1 (HMGB1), ATP and translocation of calreticulin to cell surface. In response to DNA damage, cytosolic DNA released from the nucleus mitochondria activates sensors like cGAS/STING, leading to an increase in IFNβ, that recruits and activates BAFT3+ dendritic cells, crucial agents for cross-presentation and cross-priming of CD8+ T cells. Radiation also increases the trafficking of activated CD8+ T cells by releasing CXCL16, a chemokine that binds to CXCR6 (Matsumura et al., J Immunol 181:3099-3107 2008). Recent evidence has demonstrated how as part of DNA damage response to RT, tumor mutations are expressed and can generate neoantigens that contribute to sustained immune responses in patients (Nature Medicine, 2018; 24;12:1845). Consequently, RT can recruit both the innate and adaptive immune response and, in combination with immune checkpoint blockade (ICB) can convert the irradiated tumor into an in situ vaccine. The choice of technical parameters like radiation dose rate, dose and fractionation and the type of field chosen, all impact on the success of RT and immunotherapy combinations, as demonstrated by impairment of immune response when nodal regions are included in the field treated by RT and ICB (Marciscano A et al: Clin Cancer Res: 4(20) October 15, 2018). The widespread access to radiotherapy centers and the ease to include a standard modality like RT in clinical trials have have rendered it one of the tools of modern immunotherapy of cancer, by exploiting RT effects on tumor and TME, beyond its classical cytocidal potential. Citation Format: Silvia Formenti. Radiotherapy effects on tumor microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr IA003.

Combined Photothermal and Ionizing Radiation Sensitization of Triple-Negative Breast Cancer Using Triangular Silver Nanoparticles.

BackgroundIonizing radiation (IR) is commonly used in triple-negative breast cancer (TNBC) treatment regimens. However, off-target toxicity affecting normal tissue and grueling treatment regimens remain major limitations. Hyperthermia is one of the greatest IR sensitizers, but only if heat is administered simultaneously or immediately prior to ionizing radiation. Difficulty in co-localizing ionizing radiation (IR) in rapid succession with hyperthermia, and confining treatment to the tumor have hindered widespread clinical adoption of combined thermoradiation treatment. Metal nanoparticle-based approaches to IR sensitization and photothermal heat generation may aid in overcoming these issues and improve treatment specificity.MethodsWe assessed the potential to selectively treat MDA-MB-231 TNBC cells without affecting non-malignant MCF-10A breast cells using a multimodal approach based upon combined photothermal therapy, IR sensitization, and specific cytotoxicity using triangular silver nanoparticles (TAgNPs) with peak absorbance in the near-infrared light (NIR) spectrum.ResultsWe found that TAgNP-mediated photothermal therapy and radiosensitization offer a high degree of specificity for treatment of TNBC without affecting non-malignant mammary epithelial cells.DiscussionIf given at a high enough dose, IR, heat, or TAgNPs alone could be sufficient for tumor treatment. However, when the dose of one or all of these modalities increases, off-target effects also increase. The challenge lies in identifying the minimal doses of each individual treatment such that when combined they provide maximum selectivity for treatment of TNBC cells with minimum off-target effects on non-malignant breast cells. Our results provide proof of concept that this combination is highly selective for TNBC cells while sparing non-malignant mammary epithelial cells. This treatment would be particularly important for patients undergoing breast conservation therapy and for treatment of invasive tumor margins near the periphery where each individual treatment might be at a sub-therapeutic level.

Norepinephrine transporter expressed on mammary epithelial cells incorporates norepinephrine in milk into the cells

Mammary epithelial cells synthesize and secrete norepinephrine (NE) into breast milk to regulate β-casein expression through the adrenergic β2 receptor. We investigated the expression, localization, and roles of NE transporter (NET) in the mammary epithelium during lactation. mRNA and protein levels of NET were determined in primary normal human mammary epithelial cells (pHMECs) and non-malignant human mammary epithelial MCF-12A cells. In nursing CD1 mice, NET localized to the apical membranes of the mammary epithelium. The intracellular NE content of pHMECs incubated with NE increased. Although the β-casein concentration in milk was slightly higher at day 10 than at day 2 of lactation, the NE concentration and lactation-related proteins were only slightly changed on days 2–10. Restraint stress increased the NE concentration in milk from nursing mice and NET protein levels were significantly higher than in non-stressed nursing mice. NET is expressed on the apical membrane of mammary epithelial cells and incorporates NE in milk into cells, potentially regulating the NE concentration in milk.

Deregulation of a Cis-Acting lncRNA in Non-small Cell Lung Cancer May Control HMGA1 Expression.

BackgroundLong non-coding RNAs (lncRNAs) have long been implicated in cancer-associated phenotypes. Recently, a class of lncRNAs, known as cis-acting, have been shown to regulate the expression of neighboring protein-coding genes and may represent undiscovered therapeutic action points. The chromatin architecture modification gene HMGA1 has recently been described to be aberrantly expressed in lung adenocarcinoma (LUAD). However, the mechanisms mediating the expression of HMGA1 in LUAD remain unknown. Here we investigate the deregulation of a putative cis-acting lncRNA in LUAD, and its effect on the oncogene HMGA1.MethodsLncRNA expression was determined from RNA-sequencing data of tumor and matched non-malignant tissues from 36 LUAD patients. Transcripts with significantly deregulated expression were identified and validated in a secondary LUAD RNA-seq dataset (TCGA). SiRNA-mediated knockdown of a candidate cis-acting lncRNA was performed in BEAS-2B cells. Quantitative real-time PCR was used to observe the effects of lncRNA knockdown on the expression of HMGA1.ResultsWe identified the lncRNA RP11.513I15.6, which we refer to as HMGA1-lnc, neighboring HMGA1 to be significantly downregulated in both LUAD cohorts. Conversely, we found HMGA1 significantly overexpressed in LUAD and anticorrelated with HMGA1-lnc. In vitro experiments demonstrated siRNA-mediated inhibition of HMGA1-lnc in immortalized non-malignant lung epithelial cells resulted in a significant increase in HMGA1 gene expression.ConclusionOur results suggest that HMGA1-lnc is a novel cis-acting lncRNA that negatively regulates HMGA1 gene expression in lung cells. Further characterization of this regulatory mechanism may advance our understanding of the maintenance of lung cancer phenotypes and uncover a novel therapeutic intervention point for tumors driven by HMGA1.

The exosome-like vesicles derived from androgen exposed-prostate stromal cells promote epithelial cells proliferation and epithelial-mesenchymal transition

Benign prostatic hyperplasia (BPH) is an age-related disease in men. Mesenchymal /stromal and epithelial cells interactions are essential to prostate functions. In this study, human nonmalignant prostate epithelial RWPE-1 cells were cocultured with testosterone (TE) -exposed prostate stromal fibroblasts WPMY-1 cells (TE-WPMY-1). The survival rate, epithelial-mesenchymal transition (EMT) and collagen deposition of RWPE-1 were observed. The expression profiles of circRNAs, lncRNAs and mRNAs in WPMY-1-derived exosome-like vesicles (WPMY-1-exo) were explored by high-throughput RNA sequencing. Firstly, both TE-WPMY-1 and TE-WPMY-1-exo significantly promoted RWPE-1 cells proliferation. Secondly, 41 circRNAs, 132 lncRNAs and 1057 mRNAs were differentially expressed (DE) between TE-WPMY-1-exo and the control. Functional enrichment analyses, co-expression analyses and quantitative real-time PCR verification showed that the DE RNAs played important roles in cell proliferation, structure, phenotype and fibrosis. Lastly, blocking WPMY-1-exo biogenesis/release by GW4869 can attenuate TE-WPMY-1-stimulated RWPE-1 cells EMT and collagen deposition. Taken together, our results indicated that WPMY-1-exo modulated the phenotypes changes and collagen deposition of prostate epithelial cells. It provided a novel basis for understanding the underlying mechanisms of RWPE-1 cells EMT and fibrosis induced by WPMY-1 in BPH.