Differential reprogramming of breast cancer subtypes in 3D cultures and implications for sensitivity to targeted therapy.
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ABSTRACT:
SUBMITTER: Koedoot E
PROVIDER: S-EPMC8012355 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes (OCT4, NANOG, SOX2, KLF4, CMYC, ABCG2 and PAX6) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors (OCT4, NANOG, SOX2, KLF4 and CMYC) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4, NANOG, SOX2, KLF4, HNK1, ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2, ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine.
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Project description:In solid tumors, such as breast cancer, cells are exposed to hypoxia. Cancer cells adapt their metabolism by activating hypoxia-inducible factors (HIFs) that promote the transcription of genes involved in processes such as cell survival, drug resistance and metastasis. HIF-1 is also induced in an oxygen-independent manner through the activation of epidermal growth factor receptor tyrosine kinase (EGFR-TK). Triple-negative breast cancer (TNBC) is a subtype of invasive breast cancer characterized by negative expression of hormonal and HER2 receptors, and this subtype generally overexpresses EGFR. Sensitivity to three EGFR inhibitors (cetuximab, gefitinib and lapatinib, an HER2/EGFR-TK inhibitor) was evaluated in a metastatic TNBC cell model (MDA-MB-231), and the impact of these drugs on the activity and stability of HIF was assessed.MDA-MB-231 cells were genetically modified to stably express an enhanced green fluorescent protein (EGFP) induced by hypoxia; the Ca9-GFP cell model reports HIF activity, whereas GFP-P564 reports HIF stability. The reporter signal was monitored by flow cytometry. HIF-1 DNA-binding activity, cell migration and viability were also evaluated in response to EGFR inhibitors. Cell fluorescence signals strongly increased under hypoxic conditions (> 30-fold). Cetuximab and lapatinib did not affect the signal induced by hypoxia, whereas gefitinib sharply reduced its intensity in both cell models and also diminished HIF-1 alpha levels and HIF-1 DNA-binding activity in MDA-MB-231 cells. This gefitinib feature was associated with its ability to inhibit MDA-MB-231 cell migration and to induce cell mortality in a dose-dependent manner. Cetuximab and lapatinib had no effect on cell migration or cell viability.Resistance to cetuximab and lapatinib and sensitivity to gefitinib were associated with their ability to modulate HIF activity and stability. In conclusion, downregulation of HIF-1 through EGFR signaling seems to be required for the induction of a positive response to EGFR-targeted therapies in TNBC.
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Project description:Solid tumours naturally grow in 3D wherein the spatial arrangement of cells affects how they interact with each other. This suggests that 3D cell culture may mimic the natural in vivo setting better than traditional monolayer (2D) cell culture, where cells are grown attached to plastic. Here, using HER2-positive breast cancer cell lines as models (BT474, HCC1954, EFM192A), the effects of culturing cells in 3D using the poly-HEMA method compared to 2D cultures were assessed in terms of cellular viability, response/resistance to anti-cancer drugs, protein expression and enzyme activity. Scanning electron microscopy showed the morphology of cells in 3D to be substantially different to those cultured in 2D. Cell viability in 3D cells was substantially lower than that of cells in 2D cultures, while 3D cultures were more resistant to the effects of HER-targeted (neratinib) and classical chemotherapy (docetaxel) drugs. Expression of proteins involved in cell survival, transporters associated with drug resistance and drug targets were increased in 3D cultures. Finally, activity of drug metabolising enzyme CYP3A4 was substantially increased in 3D compared to 2D cultures. Together this data indicates that the biological information represented by 3D and 2D cell cultures is substantially different i.e. 3D cell cultures demonstrate higher innate resistance to anti-cancer drugs compared to 2D cultures, which may be facilitated by the altered receptor proteins, drug transporters and metabolising enzyme activity. This highlights the importance of considering 3D in addition to 2D culture methods in pre-clinical studies of both newer targeted and more traditional anti-cancer drugs.
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Project description:Current standard-of-care (SOC) therapy for breast cancer includes targeted therapies such as endocrine therapy for estrogen receptor-alpha (ERα) positive; anti-HER2 monoclonal antibodies for human epidermal growth factor receptor-2 (HER2)-enriched; and general chemotherapy for triple negative breast cancer (TNBC) subtypes. These therapies frequently fail due to acquired or inherent resistance. Altered metabolism has been recognized as one of the major mechanisms underlying therapeutic resistance. There are several cues that dictate metabolic reprogramming that also account for the tumors' metabolic plasticity. For metabolic therapy to be efficacious there is a need to understand the metabolic underpinnings of the different subtypes of breast cancer as well as the role the SOC treatments play in targeting the metabolic phenotype. Understanding the mechanism will allow us to identify potential therapeutic vulnerabilities. There are some very interesting questions being tackled by researchers today as they pertain to altered metabolism in breast cancer. What are the metabolic differences between the different subtypes of breast cancer? Do cancer cells have a metabolic pathway preference based on the site and stage of metastasis? How do the cell-intrinsic and -extrinsic cues dictate the metabolic phenotype? How do the nucleus and mitochondria coordinately regulate metabolism? How does sensitivity or resistance to SOC affect metabolic reprogramming and vice-versa? This review addresses these issues along with the latest updates in the field of breast cancer metabolism.
| S-EPMC6406734 | biostudies-literature
Project description:Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.
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Project description:Purpose: By catalyzing hydrolysis of cyclic guanosine monophosphate, phosphodiesterase (PDE) 5 is a critical regulator of its concentration and biological effects in different (patho)physiological processes, including cancers. Being PDE5 a known druggable target, we investigated the clinical significance of its expression in breast cancers and the underlying molecular mechanisms by which it may contribute to breast cancer progression. Experimental Design: RT-PCR and immunoblotting analyses were used for PDE5 expression in eight breast cancer cell lines. To examine PDE5âs impact on cancer phenotype, MCF-7 cells, expressing the lowest levels of PDE5, were engineered to stably overexpress PDE5. Cell proliferation was assessed by MTT assays, motility and invasion by wound-healing, transmigration, matrigel-based invasion assays. RNA sequencing identified differentially-expressed genes. Clinical relevance of PDE5 was investigated by immunohistochemistry on tissues and retrospective studies from the Metabric cohort. Results: PDE5 was expressed at lower levels in luminal A-type breast cancer cells (MCF-7) compared to luminal B-like, HER2-overexpressing and basal-like cells. MCF-7 cells stably overexpressing PDE5 exhibited an increased cell motility and invasion through activation of Rho family of GTPases. Treatment of PDE5 clones with selective ROCK or PDE5 inhibitors completely restored the less motile and weak invasive behavior of control cells. In patients, PDE5 expression was found in ~85% of tumor entities analyzed, with the highest intensity staining in high-grade tumors. Retrospective analyses showed that higher PDE5 levels correlated with poorer survival. Conclusion: PDE5 expression enhances breast cancer cell invasive potential, highlighting its role as a novel molecular candidate with prognostic significance and a potential target in the treatment of breast cancers.
2016-02-01 | E-MTAB-3801 | biostudies-arrayexpress