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SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis

View Article: PubMed Central - PubMed

ABSTRACT

Although cancers are considered stem cell diseases, mechanisms involving stem cell alterations are poorly understood. Squamous cell carcinoma (SQCC) is the second most common lung cancer, and its pathogenesis appears to hinge on changes in the stem cell behavior of basal cells in the bronchial airways. Basal cells are normally quiescent and differentiate into mucociliary epithelia. Smoking triggers a hyperproliferative response resulting in progressive premalignant epithelial changes ranging from squamous metaplasia to dysplasia. These changes can regress naturally, even with chronic smoking. However, for unknown reasons, dysplasias have higher progression rates than earlier stages. We used primary human tracheobronchial basal cells to investigate how copy number gains in SOX2 and PIK3CA at 3q26-28, which co-occur in dysplasia and are observed in 94% of SQCCs, may promote progression. We find that SOX2 cooperates with PI3K signaling, which is activated by smoking, to initiate the squamous injury response in basal cells. This response involves SOX9 repression, and, accordingly, SOX2 and PI3K signaling levels are high during dysplasia, while SOX9 is not expressed. By contrast, during regeneration of mucociliary epithelia, PI3K signaling is low and basal cells transiently enter a SOX2LoSOX9Hi state, with SOX9 promoting proliferation and preventing squamous differentiation. Transient reduction in SOX2 is necessary for ciliogenesis, although SOX2 expression later rises and drives mucinous differentiation, as SOX9 levels decline. Frequent coamplification of SOX2 and PIK3CA in dysplasia may, thus, promote progression by locking basal cells in a SOX2HiSOX9Lo state with active PI3K signaling, which sustains the squamous injury response while precluding normal mucociliary differentiation. Surprisingly, we find that, although later in invasive carcinoma SOX9 is generally expressed at low levels, its expression is higher in a subset of SQCCs with less squamous identity and worse clinical outcome. We propose that early pathogenesis of most SQCCs involves stabilization of the squamous injury state in stem cells through copy number gains at 3q, with the pro-proliferative activity of SOX9 possibly being exploited in a subset of SQCCs in later stages.

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Precocious SOX2 expression in proliferating tracheobronchial basal cells enhances mucinous differentiation.(A) Experimental design. SOX2Lo tracheobronchial basal cells proliferating on plastic were infected overnight with Lenti-SOX2 or empty vector, seeded subconfluently at ALI, and examined after 5 wk. (B) Hematoxylin and eosin (H&E) staining showing ectopic induction of glandular-like areas and squamous metaplasia by Lenti-SOX2. (C) PAS-D (periodic acid Schiff-diastase) staining for mucins. Glandular differentiation was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Plotted numerical data are in S1 Data. (D) MUC16 and MUC5AC mucin staining. Arrows point to non-glandular cells with increased MUC16 expression relative to vector control cultures. Due to high MUC16 expression in Lenti-SOX2 cultures, the exposure time was shorter than in Fig 1C. (E) MUC16 and MUC5AC mucin staining in native human tracheobronchial tissue. Scale bars are 100 μm (B), 50 μm (C), and 20 μm (D, E). Significance was calculated using a two-tailed t test. *p = 0.006.
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pbio.1002581.g002: Precocious SOX2 expression in proliferating tracheobronchial basal cells enhances mucinous differentiation.(A) Experimental design. SOX2Lo tracheobronchial basal cells proliferating on plastic were infected overnight with Lenti-SOX2 or empty vector, seeded subconfluently at ALI, and examined after 5 wk. (B) Hematoxylin and eosin (H&E) staining showing ectopic induction of glandular-like areas and squamous metaplasia by Lenti-SOX2. (C) PAS-D (periodic acid Schiff-diastase) staining for mucins. Glandular differentiation was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Plotted numerical data are in S1 Data. (D) MUC16 and MUC5AC mucin staining. Arrows point to non-glandular cells with increased MUC16 expression relative to vector control cultures. Due to high MUC16 expression in Lenti-SOX2 cultures, the exposure time was shorter than in Fig 1C. (E) MUC16 and MUC5AC mucin staining in native human tracheobronchial tissue. Scale bars are 100 μm (B), 50 μm (C), and 20 μm (D, E). Significance was calculated using a two-tailed t test. *p = 0.006.

Mentions: To investigate whether precocious SOX2 expression, which would be caused by SOX2 amplification, affects mucociliary differentiation, we expressed SOX2 in proliferating basal cell ALI cultures before its normal time of expression. SOX2 was expressed from a lentiviral vector at the same mRNA levels seen in normal native tissue and SOX2-amplified SQCCs (Fig 1B, Lenti-SOX2). Proliferating basal cells were infected on plastic dishes overnight, seeded subconfluently into ALI cultures, and differentiated over 5 wk (Fig 2A). Histological analyses of the 5-wk cultures indicated that precocious SOX2 expression enhanced mucinous differentiation and induced squamous metaplasia (Fig 2B). Lenti-SOX2 induced ectopic gland-like structures that were confirmed mucinous by periodic acid-Schiff (PAS) reactivity (Fig 2C). It also increased MUC16 expression relative to control cultures (Fig 2D), wherein endogenous SOX2 expression was naturally correlated with MUC16 expression (Fig 1C and 1D). Although MUC16+ cells have been noted in the tracheal epithelium [67], we now show that these cells do not express the canonical goblet cell marker MUC5AC (Fig 2D and 2E) and, hence, are a distinct subtype of mucinous lineage that is induced by SOX2.


SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis
Precocious SOX2 expression in proliferating tracheobronchial basal cells enhances mucinous differentiation.(A) Experimental design. SOX2Lo tracheobronchial basal cells proliferating on plastic were infected overnight with Lenti-SOX2 or empty vector, seeded subconfluently at ALI, and examined after 5 wk. (B) Hematoxylin and eosin (H&E) staining showing ectopic induction of glandular-like areas and squamous metaplasia by Lenti-SOX2. (C) PAS-D (periodic acid Schiff-diastase) staining for mucins. Glandular differentiation was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Plotted numerical data are in S1 Data. (D) MUC16 and MUC5AC mucin staining. Arrows point to non-glandular cells with increased MUC16 expression relative to vector control cultures. Due to high MUC16 expression in Lenti-SOX2 cultures, the exposure time was shorter than in Fig 1C. (E) MUC16 and MUC5AC mucin staining in native human tracheobronchial tissue. Scale bars are 100 μm (B), 50 μm (C), and 20 μm (D, E). Significance was calculated using a two-tailed t test. *p = 0.006.
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getmorefigures.php?uid=PMC5120804&req=5

pbio.1002581.g002: Precocious SOX2 expression in proliferating tracheobronchial basal cells enhances mucinous differentiation.(A) Experimental design. SOX2Lo tracheobronchial basal cells proliferating on plastic were infected overnight with Lenti-SOX2 or empty vector, seeded subconfluently at ALI, and examined after 5 wk. (B) Hematoxylin and eosin (H&E) staining showing ectopic induction of glandular-like areas and squamous metaplasia by Lenti-SOX2. (C) PAS-D (periodic acid Schiff-diastase) staining for mucins. Glandular differentiation was quantified by scoring 6–7 cm of epithelia from multiple sections derived from three replicates. Mean ± SEM is shown. Plotted numerical data are in S1 Data. (D) MUC16 and MUC5AC mucin staining. Arrows point to non-glandular cells with increased MUC16 expression relative to vector control cultures. Due to high MUC16 expression in Lenti-SOX2 cultures, the exposure time was shorter than in Fig 1C. (E) MUC16 and MUC5AC mucin staining in native human tracheobronchial tissue. Scale bars are 100 μm (B), 50 μm (C), and 20 μm (D, E). Significance was calculated using a two-tailed t test. *p = 0.006.
Mentions: To investigate whether precocious SOX2 expression, which would be caused by SOX2 amplification, affects mucociliary differentiation, we expressed SOX2 in proliferating basal cell ALI cultures before its normal time of expression. SOX2 was expressed from a lentiviral vector at the same mRNA levels seen in normal native tissue and SOX2-amplified SQCCs (Fig 1B, Lenti-SOX2). Proliferating basal cells were infected on plastic dishes overnight, seeded subconfluently into ALI cultures, and differentiated over 5 wk (Fig 2A). Histological analyses of the 5-wk cultures indicated that precocious SOX2 expression enhanced mucinous differentiation and induced squamous metaplasia (Fig 2B). Lenti-SOX2 induced ectopic gland-like structures that were confirmed mucinous by periodic acid-Schiff (PAS) reactivity (Fig 2C). It also increased MUC16 expression relative to control cultures (Fig 2D), wherein endogenous SOX2 expression was naturally correlated with MUC16 expression (Fig 1C and 1D). Although MUC16+ cells have been noted in the tracheal epithelium [67], we now show that these cells do not express the canonical goblet cell marker MUC5AC (Fig 2D and 2E) and, hence, are a distinct subtype of mucinous lineage that is induced by SOX2.

View Article: PubMed Central - PubMed

ABSTRACT

Although cancers are considered stem cell diseases, mechanisms involving stem cell alterations are poorly understood. Squamous cell carcinoma (SQCC) is the second most common lung cancer, and its pathogenesis appears to hinge on changes in the stem cell behavior of basal cells in the bronchial airways. Basal cells are normally quiescent and differentiate into mucociliary epithelia. Smoking triggers a hyperproliferative response resulting in progressive premalignant epithelial changes ranging from squamous metaplasia to dysplasia. These changes can regress naturally, even with chronic smoking. However, for unknown reasons, dysplasias have higher progression rates than earlier stages. We used primary human tracheobronchial basal cells to investigate how copy number gains in SOX2 and PIK3CA at 3q26-28, which co-occur in dysplasia and are observed in 94% of SQCCs, may promote progression. We find that SOX2 cooperates with PI3K signaling, which is activated by smoking, to initiate the squamous injury response in basal cells. This response involves SOX9 repression, and, accordingly, SOX2 and PI3K signaling levels are high during dysplasia, while SOX9 is not expressed. By contrast, during regeneration of mucociliary epithelia, PI3K signaling is low and basal cells transiently enter a SOX2LoSOX9Hi state, with SOX9 promoting proliferation and preventing squamous differentiation. Transient reduction in SOX2 is necessary for ciliogenesis, although SOX2 expression later rises and drives mucinous differentiation, as SOX9 levels decline. Frequent coamplification of SOX2 and PIK3CA in dysplasia may, thus, promote progression by locking basal cells in a SOX2HiSOX9Lo state with active PI3K signaling, which sustains the squamous injury response while precluding normal mucociliary differentiation. Surprisingly, we find that, although later in invasive carcinoma SOX9 is generally expressed at low levels, its expression is higher in a subset of SQCCs with less squamous identity and worse clinical outcome. We propose that early pathogenesis of most SQCCs involves stabilization of the squamous injury state in stem cells through copy number gains at 3q, with the pro-proliferative activity of SOX9 possibly being exploited in a subset of SQCCs in later stages.

No MeSH data available.


Related in: MedlinePlus