Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells

Vaapil, Marica; Helczynska, Karolina; Villadsen, René; Petersen, Ole W.; Johansson, Erika; Beckman, Siv; Larsson, Christer; Påhlman, Sven; Jögi, Annika

doi:10.1371/journal.pone.0046543

Cited by 36 publications

(40 citation statements)

References 50 publications

(77 reference statements)

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“…Moreover, basal cell phenotypic markers such as α6 integrin and p75 NTR (Rezvani et al, 2011; Nakamura et al, 2007) were noted to be expressed in cells under 0.5% oxygen pressure. This was in agreement with previous studies on skin keratinocytes (Rezvani et al, 2011; Ngo et al, 2007) as well as other epithelial cells (Vaapil et al, 2012; O’Callaghan et al, 2011). Taken together, these outcomes illustrate that hypoxia can maintain oral keratinocytes in an undifferentiated state.…”

Section: Discussionsupporting

confidence: 94%

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

Kato

Izumi

Uenoyama

et al. 2014

Cells Tissues Organs

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The aim of this study was to determine the effects of hypoxia on the proliferating potential and phenotype of primary human oral keratinocytes cultured at ambient oxygen tension (20%) or at different levels of hypoxia (2 and 0.5% O₂). The effects of oxygen tensions on cellular metabolic activity, cell proliferation, clonogenicity and proliferation heterogeneity were measured. Cell cycle profiles were analyzed by a fluorescent-activated cell sorter, and p21^WAF1/CIP1 expression in the G₀/G₁ phase was also concomitantly quantitated. The expression levels of cell cycle regulatory proteins were examined by immunoblotting, and the cellular senescence was assessed by senescence-associated β-galactosidase staining. Basal and suprabasal keratinocyte phenotypes were determined by the expression levels of 14-3-3σ, p75^NTR and α6 integrin. Despite having a lower metabolism, the proliferation rate and clonogenic potential were remarkably enhanced in hypoxic cells. The significantly higher percentage of cells in the G₀/G₁ phase under hypoxia and the expression patterns of cell cycle regulatory proteins in hypoxic cells were indicative of a state of cell cycle arrest in hypoxia. Furthermore, a decrease in the expression of p21^WAF1/CIP1 and p16^INK4A and fewer β-galactosidase-positive cells suggested a quiescent phenotype rather than a senescent one in hypoxic cells. Compared with normoxic cells, the differential expression patterns of keratinocyte phenotypic markers suggest that hypoxic cells that generate minimal reactive oxygen species, suppress the mammalian target of rapamycin activity and express hypoxia-inducible factor-1α favor a basal cell phenotype. Thus, regardless of the predisposition to the state of cell cycle arrest, hypoxic conditions can maintain oral keratinocytes in vitro in an undifferentiated and quiescent state.

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Section: Discussionsupporting

confidence: 94%

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

Kato

Izumi

Uenoyama

et al. 2014

Cells Tissues Organs

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“…Physiological hypoxia induces aggressiveness and metastatic phenotype in normal breast epithelial cells (22). Hif-1α is a master regulator of various signaling pathways and known to play an important role in tumor progression and angiogenesis in solid tumors (40).…”

Section: Discussionmentioning

confidence: 99%

“…To test this hypothesis, we developed first of its kind in vitro human tumor progression model using microfabrication to generate uniform and defined size microtumors of various breast cancer cell lines (MCF7, T47D, BT474, MDA-MB-231 and HCC1187). We could link breast tumor size to aggressive and migratory behavior by precisely modulating just the microtumor size and without any exposure to artificial hypoxic chambers or chemical stimuli (22). Our results demonstrated that large size (600 μm) microtumors of non-invasive estrogen/progesterone receptor positive (ER+/PR+) breast tumor cells (MCF7, T47D) upregulate mesenchymal markers and ‘migrate out’ from the hydrogel microwells without losing their epithelial phenotype.…”

Section: Introductionmentioning

confidence: 99%

Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression

et al. 2016

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Tumor size is strongly correlated with breast cancer metastasis and patient survival. Increased tumor size contributes to hypoxic and metabolic gradients in the solid tumor and to an aggressive tumor phenotype. Thus, it is important to develop three-dimensional (3D) breast tumor models that recapitulate size-induced microenvironmental changes and consequently, natural tumor progression in real time without the use of artificial culture conditions or gene manipulations. Here, we developed size-controlled multicellular aggregates (“microtumors”) of subtype-specific breast cancer cells by using non-adhesive polyethylene glycol dimethacrylate hydrogel microwells of defined sizes (150–600 μm). These 3D microtumor models faithfully represent size-induced microenvironmental changes such as hypoxic gradients, cellular heterogeneity and spatial distribution of necrotic/proliferating cells. These microtumors acquire hallmarks of tumor progression in the same cell lines within 6 days. Of note, large microtumors of hormone receptor positive cells exhibited an aggressive phenotype characterized by collective cell migration and upregulation of mesenchymal markers at mRNA and protein level, which was not observed in small microtumors. Interestingly, triple negative breast cancer (TNBC) cell lines did not show size-dependent upregulation of mesenchymal markers. In conclusion, size-controlled microtumor models successfully recapitulated clinically observed positive association between tumor size and aggressive phenotype in hormone receptor positive breast cancer while maintaining clinically proven poor correlation of tumor size with aggressive phenotype in TNBC. Such clinically relevant 3D models generated under controlled experimental conditions can serve as precise preclinical models to study mechanisms involved in breast tumor progression as well as antitumor drug effects as a function of tumor progression.

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“…1,3,4 In cancer, hypoxia is an important contributor to carcinogenesis 5 and cancer phenotype. 6,7 Factors contributing to an imbalance of oxygen demand and supply lead to adaptive cellular mechanisms being initiated and involving vast transcriptional and post-transcriptional changes in gene expression, of which the hypoxia-inducible factor 1 (HIF1) is a master regulator. 8 HIF1 is composed of two subunits; the HIF1a (HIF1A) and the aryl hydrocarbon receptor nuclear translator (ARNT), with HIF1A induced and continuously degraded via an association with the von Hippel-Lindau protein ubiquitin E3 ligase complex leading to degradation via the ubiquitinproteosome pathway in normoxia.…”

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confidence: 99%

Nuclear HIF1A expression is strongly prognostic in sporadic but not familial male breast cancer

et al. 2014

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Male breast cancer is poorly understood with a large proportion arising in the familial context particularly with the BRCA2 germline mutation. As phenotypic and genotypic differences between sporadic and familial male breast cancers have been noted, we investigated the importance of a hypoxic drive in these cancers as this pathway has been shown to be of importance in familial female breast cancer. Expression of two major hypoxiainduced proteins, the hypoxia-inducible factor-1a (HIF1A) and the carbonic anhydrase IX (CA9), examined within a large cohort including 61 familial (3 BRCA1, 28 BRCA2, 30 BRCAX) and 225 sporadic male breast cancers showed that 31% of all male breast cancers expressed either HIF1A (25%) and/or CA9 (8%) in the combined cohort. Expression of HIF1A correlated with an increased incidence of a second-major malignancy (P ¼ 0.04), histological tumor type (P ¼ 0.005) and basal phenotype (P ¼ 0.02). Expression of CA9 correlated with age (P ¼ 0.004) in sporadic cases and an increased tumor size (P ¼ 0.003). Expression of HIF1A was prognostic for disease-specific survival in sporadic male breast cancers (HR: 3.8, 95% CI: 1.5-9.8, P ¼ 0.006) but not within familial male breast cancer, whereas CA9 was only prognostic in familial male breast cancers (HR: 358.0, 95% CI: 9.3-13781.7, P ¼ 0.002) and not in sporadic male breast cancer. This study found that hypoxic drive is less prevalent in male breast cancer compared with female breast cancer, possibly due to a different breast microenvironment. The prognostic impact of HIF1A is greatest in sporadic male breast cancers with an alternate dominant mechanism for the oncogenic drivers suggested in high risk familial male breast cancers.

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Hypoxic Conditions Induce a Cancer-Like Phenotype in Human Breast Epithelial Cells

Cited by 36 publications

References 50 publications

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

Hypoxia Induces an Undifferentiated Phenotype of Oral Keratinocytes in vitro

Three-Dimensional Breast Cancer Models Mimic Hallmarks of Size-Induced Tumor Progression

Nuclear HIF1A expression is strongly prognostic in sporadic but not familial male breast cancer

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