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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.

No MeSH data available.


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During mucociliary differentiation of tracheobronchial basal cells, SOX2 expression varies from low to high.(A) SOX2 immunohistochemistry (IHC) in normal native human tracheobronchial epithelia and SOX2-amplified primary patient lung SQCCs and SQCC patient-derived xenografts (PDXs). Arrows point to some basal cells. (B) qRT-PCR quantification of SOX2 expression in normal tracheobronchial epithelial cells and SQCCs. Tracheobronchial cell suspensions and FACS-purified basal cells from these suspensions were derived from tissue without culturing. ADC = primary patient lung adenocarcinoma. All data, with the exception of “Proliferating basal cells on plastic” (green), are from individual biological replicates, which were generated from duplicate qRT-PCR technical replicates. Tracheobronchial basal cells proliferating on plastic were infected with Lenti-SOX2 or empty vector, with mean expression ± standard error of the mean (SEM) from three biological replicate experiments shown. Control empty vector did not alter SOX2 expression relative to untransduced basal cells (not shown). LRR = log likelihood ratio quantification of SOX2 gene copy number. Expression is normalized to normal tracheal suspension #1, which was assigned a value of 100. (C, D) SOX2 and mucociliary lineage marker expression during tracheobronchial basal cell differentiation in air-liquid-interface (ALI) cultures. (C) Immunofluorescence staining of SOX2 and lineage marker expression. White arrows point to some basal cells and green arrows mark FOXJ1+ cells. (D) qRT-PCR analysis of SOX2 and lineage marker expression. Data are plotted relative to the time point with the maximal expression of the gene, which was given a value of 100. Means ± SEM from three replicates are shown. (E) SOX2 IHC in metaplastic areas of native human tracheobronchial epithelia. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
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pbio.1002581.g001: During mucociliary differentiation of tracheobronchial basal cells, SOX2 expression varies from low to high.(A) SOX2 immunohistochemistry (IHC) in normal native human tracheobronchial epithelia and SOX2-amplified primary patient lung SQCCs and SQCC patient-derived xenografts (PDXs). Arrows point to some basal cells. (B) qRT-PCR quantification of SOX2 expression in normal tracheobronchial epithelial cells and SQCCs. Tracheobronchial cell suspensions and FACS-purified basal cells from these suspensions were derived from tissue without culturing. ADC = primary patient lung adenocarcinoma. All data, with the exception of “Proliferating basal cells on plastic” (green), are from individual biological replicates, which were generated from duplicate qRT-PCR technical replicates. Tracheobronchial basal cells proliferating on plastic were infected with Lenti-SOX2 or empty vector, with mean expression ± standard error of the mean (SEM) from three biological replicate experiments shown. Control empty vector did not alter SOX2 expression relative to untransduced basal cells (not shown). LRR = log likelihood ratio quantification of SOX2 gene copy number. Expression is normalized to normal tracheal suspension #1, which was assigned a value of 100. (C, D) SOX2 and mucociliary lineage marker expression during tracheobronchial basal cell differentiation in air-liquid-interface (ALI) cultures. (C) Immunofluorescence staining of SOX2 and lineage marker expression. White arrows point to some basal cells and green arrows mark FOXJ1+ cells. (D) qRT-PCR analysis of SOX2 and lineage marker expression. Data are plotted relative to the time point with the maximal expression of the gene, which was given a value of 100. Means ± SEM from three replicates are shown. (E) SOX2 IHC in metaplastic areas of native human tracheobronchial epithelia. Scale bars are 20 μm. All plotted numerical data are in S1 Data.

Mentions: In SQCCs, SOX2 copy number gains are correlated with increased SOX2 expression (S1A Fig) [41,42,63]. However, the extent to which SOX2 is overexpressed relative to normal basal cells has not been reported. Surprisingly, we found that SOX2-amplified SQCC primary patient tumors and patient-derived xenografts (PDXs) expressed similar levels of SOX2 protein as normal cells in native human tracheobronchial epithelia, including basal cells (Figs 1A and S1B). In support of this finding, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that freshly harvested cell suspensions from tracheobronchial tissue, as well as FACS-purified basal cells from these suspensions, expressed equivalent levels of SOX2 mRNA as SOX2-amplified SQCC PDXs (Fig 1B). These normal levels of expression were also much higher than in non-SOX2-amplified SQCC and ADC PDXs (Fig 1B).


SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis
During mucociliary differentiation of tracheobronchial basal cells, SOX2 expression varies from low to high.(A) SOX2 immunohistochemistry (IHC) in normal native human tracheobronchial epithelia and SOX2-amplified primary patient lung SQCCs and SQCC patient-derived xenografts (PDXs). Arrows point to some basal cells. (B) qRT-PCR quantification of SOX2 expression in normal tracheobronchial epithelial cells and SQCCs. Tracheobronchial cell suspensions and FACS-purified basal cells from these suspensions were derived from tissue without culturing. ADC = primary patient lung adenocarcinoma. All data, with the exception of “Proliferating basal cells on plastic” (green), are from individual biological replicates, which were generated from duplicate qRT-PCR technical replicates. Tracheobronchial basal cells proliferating on plastic were infected with Lenti-SOX2 or empty vector, with mean expression ± standard error of the mean (SEM) from three biological replicate experiments shown. Control empty vector did not alter SOX2 expression relative to untransduced basal cells (not shown). LRR = log likelihood ratio quantification of SOX2 gene copy number. Expression is normalized to normal tracheal suspension #1, which was assigned a value of 100. (C, D) SOX2 and mucociliary lineage marker expression during tracheobronchial basal cell differentiation in air-liquid-interface (ALI) cultures. (C) Immunofluorescence staining of SOX2 and lineage marker expression. White arrows point to some basal cells and green arrows mark FOXJ1+ cells. (D) qRT-PCR analysis of SOX2 and lineage marker expression. Data are plotted relative to the time point with the maximal expression of the gene, which was given a value of 100. Means ± SEM from three replicates are shown. (E) SOX2 IHC in metaplastic areas of native human tracheobronchial epithelia. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
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pbio.1002581.g001: During mucociliary differentiation of tracheobronchial basal cells, SOX2 expression varies from low to high.(A) SOX2 immunohistochemistry (IHC) in normal native human tracheobronchial epithelia and SOX2-amplified primary patient lung SQCCs and SQCC patient-derived xenografts (PDXs). Arrows point to some basal cells. (B) qRT-PCR quantification of SOX2 expression in normal tracheobronchial epithelial cells and SQCCs. Tracheobronchial cell suspensions and FACS-purified basal cells from these suspensions were derived from tissue without culturing. ADC = primary patient lung adenocarcinoma. All data, with the exception of “Proliferating basal cells on plastic” (green), are from individual biological replicates, which were generated from duplicate qRT-PCR technical replicates. Tracheobronchial basal cells proliferating on plastic were infected with Lenti-SOX2 or empty vector, with mean expression ± standard error of the mean (SEM) from three biological replicate experiments shown. Control empty vector did not alter SOX2 expression relative to untransduced basal cells (not shown). LRR = log likelihood ratio quantification of SOX2 gene copy number. Expression is normalized to normal tracheal suspension #1, which was assigned a value of 100. (C, D) SOX2 and mucociliary lineage marker expression during tracheobronchial basal cell differentiation in air-liquid-interface (ALI) cultures. (C) Immunofluorescence staining of SOX2 and lineage marker expression. White arrows point to some basal cells and green arrows mark FOXJ1+ cells. (D) qRT-PCR analysis of SOX2 and lineage marker expression. Data are plotted relative to the time point with the maximal expression of the gene, which was given a value of 100. Means ± SEM from three replicates are shown. (E) SOX2 IHC in metaplastic areas of native human tracheobronchial epithelia. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
Mentions: In SQCCs, SOX2 copy number gains are correlated with increased SOX2 expression (S1A Fig) [41,42,63]. However, the extent to which SOX2 is overexpressed relative to normal basal cells has not been reported. Surprisingly, we found that SOX2-amplified SQCC primary patient tumors and patient-derived xenografts (PDXs) expressed similar levels of SOX2 protein as normal cells in native human tracheobronchial epithelia, including basal cells (Figs 1A and S1B). In support of this finding, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis indicated that freshly harvested cell suspensions from tracheobronchial tissue, as well as FACS-purified basal cells from these suspensions, expressed equivalent levels of SOX2 mRNA as SOX2-amplified SQCC PDXs (Fig 1B). These normal levels of expression were also much higher than in non-SOX2-amplified SQCC and ADC PDXs (Fig 1B).

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