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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.


Related in: MedlinePlus

SOX9 promotes basal cell proliferation and inhibits squamous differentiation.(A) Elevation of SOX9 expression in tracheobronchial basal cells promotes growth and induces MUC16 expression. Basal cells proliferating on plastic were infected with Lenti-SOX9 or control vector. The red color in all of the schematics indicates Lenti-SOX2-transduced cells. For growth assays, 5 d after infection, cells were replated at low density and cell number quantified after 7 d by alamarBlue. Data are normalized to the amount of growth in control vector cultures, which was given a value of 100. Means ± SEM from quadruplicate cultures are shown. Significance was calculated using a two-tailed t test. ***p = 0.0000005. For lineage marker expression analysis, mRNA was isolated 5 d following Lenti-SOX9 infection and quantified by qRT-PCR. Data are normalized to expression in control vector cultures, which was given a value of 1. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.02. (B) shSOX9 spontaneously increases expression of squamous markers in plastic cultures of tracheobronchial basal cells. Basal cells growing on plastic were infected with shlacz or shSOX9; after 5 d, lineage marker expression was measured by qRT-PCR. Data are plotted relative to expression in shlacz control cultures, which was given a value of 1. Means ± SEM from three replicates are shown. Significance was calculated using two-tailed t tests. *p = 0.05 (SOX9), 0.03 (SOX2), 0.02 (TMPRSS11B), 0.01 (IVL), 0.003 (SPRR1A). (C) Constitutive SOX9 expression suppresses SOX2-induced squamous differentiation in plastic cultures of tracheobronchial basal cells. Basal cells were infected with empty vector, Lenti-SOX2 alone, or Lenti-SOX2 + Lenti-SOX9; after 5 d, lineage marker expression was measured by qRT-PCR and analyzed as described in Fig 5B. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. The fold-inductions were then compared between matched pairs of Lenti-SOX2 and Lenti-SOX2 + Lenti-SOX9 coinfected cultures. The amount of marker induction in coinfected cultures was then plotted as a percentage of the induction observed with Lenti-SOX2 alone, which was given a value of 100. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. **p = 0.0006 (IVL), 0.002 (SPRR3), ***p = 0.0002 (TMPRSS11B), 0.00004 (SPRR1A). (D) Constitutive SOX9 expression suppresses SOX2-induced histologic squamous metaplasia in tracheobronchial basal cell cultures grown at ALI. Basal cells were infected with control vector or Lenti-SOX2 ± Lenti-SOX9 and grown at ALI for 5 wk. Squamous differentiation was quantified by scoring 10 cm of epithelium per replicate. Significance was calculated using a two-tailed t test relative to Lenti-SOX2 alone. ***p = 0.000001. MUC16 expression was not affected by Lenti-SOX9 coinfection with Lenti-SOX2, as assessed by immunostaining. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
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pbio.1002581.g010: SOX9 promotes basal cell proliferation and inhibits squamous differentiation.(A) Elevation of SOX9 expression in tracheobronchial basal cells promotes growth and induces MUC16 expression. Basal cells proliferating on plastic were infected with Lenti-SOX9 or control vector. The red color in all of the schematics indicates Lenti-SOX2-transduced cells. For growth assays, 5 d after infection, cells were replated at low density and cell number quantified after 7 d by alamarBlue. Data are normalized to the amount of growth in control vector cultures, which was given a value of 100. Means ± SEM from quadruplicate cultures are shown. Significance was calculated using a two-tailed t test. ***p = 0.0000005. For lineage marker expression analysis, mRNA was isolated 5 d following Lenti-SOX9 infection and quantified by qRT-PCR. Data are normalized to expression in control vector cultures, which was given a value of 1. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.02. (B) shSOX9 spontaneously increases expression of squamous markers in plastic cultures of tracheobronchial basal cells. Basal cells growing on plastic were infected with shlacz or shSOX9; after 5 d, lineage marker expression was measured by qRT-PCR. Data are plotted relative to expression in shlacz control cultures, which was given a value of 1. Means ± SEM from three replicates are shown. Significance was calculated using two-tailed t tests. *p = 0.05 (SOX9), 0.03 (SOX2), 0.02 (TMPRSS11B), 0.01 (IVL), 0.003 (SPRR1A). (C) Constitutive SOX9 expression suppresses SOX2-induced squamous differentiation in plastic cultures of tracheobronchial basal cells. Basal cells were infected with empty vector, Lenti-SOX2 alone, or Lenti-SOX2 + Lenti-SOX9; after 5 d, lineage marker expression was measured by qRT-PCR and analyzed as described in Fig 5B. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. The fold-inductions were then compared between matched pairs of Lenti-SOX2 and Lenti-SOX2 + Lenti-SOX9 coinfected cultures. The amount of marker induction in coinfected cultures was then plotted as a percentage of the induction observed with Lenti-SOX2 alone, which was given a value of 100. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. **p = 0.0006 (IVL), 0.002 (SPRR3), ***p = 0.0002 (TMPRSS11B), 0.00004 (SPRR1A). (D) Constitutive SOX9 expression suppresses SOX2-induced histologic squamous metaplasia in tracheobronchial basal cell cultures grown at ALI. Basal cells were infected with control vector or Lenti-SOX2 ± Lenti-SOX9 and grown at ALI for 5 wk. Squamous differentiation was quantified by scoring 10 cm of epithelium per replicate. Significance was calculated using a two-tailed t test relative to Lenti-SOX2 alone. ***p = 0.000001. MUC16 expression was not affected by Lenti-SOX9 coinfection with Lenti-SOX2, as assessed by immunostaining. Scale bars are 20 μm. All plotted numerical data are in S1 Data.

Mentions: Given that SOX9 expression is highest during the earliest stages of mucociliary differentiation, SOX9 may affect pre-differentiation properties of stem cells such as proliferation and lineage commitment. To determine how SOX9 affects the stem cell behavior of tracheobronchial basal cells, we first used a constitutively expressing lentiviral vector to raise its expression in plastic cultures of SOX2Lo basal cells. Lenti-SOX9 increased basal cell growth while elevating expression of SOX2 and MUC16, but not markers of squamous differentiation (Fig 10A). The increase in SOX2 mRNA expression did not translate to a detectable increase in SOX2 protein levels (S6 Fig), and the MUC16 induction was generally weaker than observed with Lenti-SOX2. These data suggest that SOX9 may have a role in promoting columnar lineage commitment, which is associated with a limited ability to increase MUC16 mRNA expression. Analysis of the effects of reduced SOX9 expression was problematic, as SOX9 shRNA lentiviruses suppressed basal cell growth. However, in short-term plastic cultures, partial SOX9 knockdown increased squamous marker, but not MUC16 expression (Fig 10B). The amount of squamous marker induction was also generally weaker than observed for Lenti-SOX2, supporting the concept that SOX9 is an early determinant of columnar versus squamous lineage commitment, rather than a strong inducer of differentiation.


SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis
SOX9 promotes basal cell proliferation and inhibits squamous differentiation.(A) Elevation of SOX9 expression in tracheobronchial basal cells promotes growth and induces MUC16 expression. Basal cells proliferating on plastic were infected with Lenti-SOX9 or control vector. The red color in all of the schematics indicates Lenti-SOX2-transduced cells. For growth assays, 5 d after infection, cells were replated at low density and cell number quantified after 7 d by alamarBlue. Data are normalized to the amount of growth in control vector cultures, which was given a value of 100. Means ± SEM from quadruplicate cultures are shown. Significance was calculated using a two-tailed t test. ***p = 0.0000005. For lineage marker expression analysis, mRNA was isolated 5 d following Lenti-SOX9 infection and quantified by qRT-PCR. Data are normalized to expression in control vector cultures, which was given a value of 1. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.02. (B) shSOX9 spontaneously increases expression of squamous markers in plastic cultures of tracheobronchial basal cells. Basal cells growing on plastic were infected with shlacz or shSOX9; after 5 d, lineage marker expression was measured by qRT-PCR. Data are plotted relative to expression in shlacz control cultures, which was given a value of 1. Means ± SEM from three replicates are shown. Significance was calculated using two-tailed t tests. *p = 0.05 (SOX9), 0.03 (SOX2), 0.02 (TMPRSS11B), 0.01 (IVL), 0.003 (SPRR1A). (C) Constitutive SOX9 expression suppresses SOX2-induced squamous differentiation in plastic cultures of tracheobronchial basal cells. Basal cells were infected with empty vector, Lenti-SOX2 alone, or Lenti-SOX2 + Lenti-SOX9; after 5 d, lineage marker expression was measured by qRT-PCR and analyzed as described in Fig 5B. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. The fold-inductions were then compared between matched pairs of Lenti-SOX2 and Lenti-SOX2 + Lenti-SOX9 coinfected cultures. The amount of marker induction in coinfected cultures was then plotted as a percentage of the induction observed with Lenti-SOX2 alone, which was given a value of 100. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. **p = 0.0006 (IVL), 0.002 (SPRR3), ***p = 0.0002 (TMPRSS11B), 0.00004 (SPRR1A). (D) Constitutive SOX9 expression suppresses SOX2-induced histologic squamous metaplasia in tracheobronchial basal cell cultures grown at ALI. Basal cells were infected with control vector or Lenti-SOX2 ± Lenti-SOX9 and grown at ALI for 5 wk. Squamous differentiation was quantified by scoring 10 cm of epithelium per replicate. Significance was calculated using a two-tailed t test relative to Lenti-SOX2 alone. ***p = 0.000001. MUC16 expression was not affected by Lenti-SOX9 coinfection with Lenti-SOX2, as assessed by immunostaining. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
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pbio.1002581.g010: SOX9 promotes basal cell proliferation and inhibits squamous differentiation.(A) Elevation of SOX9 expression in tracheobronchial basal cells promotes growth and induces MUC16 expression. Basal cells proliferating on plastic were infected with Lenti-SOX9 or control vector. The red color in all of the schematics indicates Lenti-SOX2-transduced cells. For growth assays, 5 d after infection, cells were replated at low density and cell number quantified after 7 d by alamarBlue. Data are normalized to the amount of growth in control vector cultures, which was given a value of 100. Means ± SEM from quadruplicate cultures are shown. Significance was calculated using a two-tailed t test. ***p = 0.0000005. For lineage marker expression analysis, mRNA was isolated 5 d following Lenti-SOX9 infection and quantified by qRT-PCR. Data are normalized to expression in control vector cultures, which was given a value of 1. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.02. (B) shSOX9 spontaneously increases expression of squamous markers in plastic cultures of tracheobronchial basal cells. Basal cells growing on plastic were infected with shlacz or shSOX9; after 5 d, lineage marker expression was measured by qRT-PCR. Data are plotted relative to expression in shlacz control cultures, which was given a value of 1. Means ± SEM from three replicates are shown. Significance was calculated using two-tailed t tests. *p = 0.05 (SOX9), 0.03 (SOX2), 0.02 (TMPRSS11B), 0.01 (IVL), 0.003 (SPRR1A). (C) Constitutive SOX9 expression suppresses SOX2-induced squamous differentiation in plastic cultures of tracheobronchial basal cells. Basal cells were infected with empty vector, Lenti-SOX2 alone, or Lenti-SOX2 + Lenti-SOX9; after 5 d, lineage marker expression was measured by qRT-PCR and analyzed as described in Fig 5B. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. The fold-inductions were then compared between matched pairs of Lenti-SOX2 and Lenti-SOX2 + Lenti-SOX9 coinfected cultures. The amount of marker induction in coinfected cultures was then plotted as a percentage of the induction observed with Lenti-SOX2 alone, which was given a value of 100. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. **p = 0.0006 (IVL), 0.002 (SPRR3), ***p = 0.0002 (TMPRSS11B), 0.00004 (SPRR1A). (D) Constitutive SOX9 expression suppresses SOX2-induced histologic squamous metaplasia in tracheobronchial basal cell cultures grown at ALI. Basal cells were infected with control vector or Lenti-SOX2 ± Lenti-SOX9 and grown at ALI for 5 wk. Squamous differentiation was quantified by scoring 10 cm of epithelium per replicate. Significance was calculated using a two-tailed t test relative to Lenti-SOX2 alone. ***p = 0.000001. MUC16 expression was not affected by Lenti-SOX9 coinfection with Lenti-SOX2, as assessed by immunostaining. Scale bars are 20 μm. All plotted numerical data are in S1 Data.
Mentions: Given that SOX9 expression is highest during the earliest stages of mucociliary differentiation, SOX9 may affect pre-differentiation properties of stem cells such as proliferation and lineage commitment. To determine how SOX9 affects the stem cell behavior of tracheobronchial basal cells, we first used a constitutively expressing lentiviral vector to raise its expression in plastic cultures of SOX2Lo basal cells. Lenti-SOX9 increased basal cell growth while elevating expression of SOX2 and MUC16, but not markers of squamous differentiation (Fig 10A). The increase in SOX2 mRNA expression did not translate to a detectable increase in SOX2 protein levels (S6 Fig), and the MUC16 induction was generally weaker than observed with Lenti-SOX2. These data suggest that SOX9 may have a role in promoting columnar lineage commitment, which is associated with a limited ability to increase MUC16 mRNA expression. Analysis of the effects of reduced SOX9 expression was problematic, as SOX9 shRNA lentiviruses suppressed basal cell growth. However, in short-term plastic cultures, partial SOX9 knockdown increased squamous marker, but not MUC16 expression (Fig 10B). The amount of squamous marker induction was also generally weaker than observed for Lenti-SOX2, supporting the concept that SOX9 is an early determinant of columnar versus squamous lineage commitment, rather than a strong inducer of differentiation.

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