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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

PI3K signaling is necessary for the squamous response to SOX2.(A) The PI3K inhibitor BKM120 inhibits growth of tracheobronchial basal cells. Basal cells were grown on plastic over 7 d with fresh media and drug added every other day. Growth was quantified by alamarBlue and is normalized to control cells treated with the DMSO vehicle, which was given a value of 100%. The means ± SEM from three replicates are shown. (B-E) Inhibition of PI3K signaling prevents SOX2-driven squamous differentiation. Tracheobronchial basal cells growing on plastic were infected with Lenti-SOX2 or empty vector, co-treated with the indicated drugs or shRNA viruses for 5 d, and then assayed for lineage marker expression by qRT-PCR. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. Because the magnitudes of inductions sometimes varied between biological replicate experiments, fold-inductions were directly compared between matched pairs of Lenti-SOX2 (with DMSO or shluc) and PI3K-inhibited (chemical inhibitor or shPIK3CA) Lenti-SOX2-transduced cultures. The amount of marker induction in inhibitor-treated cultures was then plotted as a percentage of the induction response seen without inhibitor treatment, which was given a value of 100. (B) Schematic for the PI3K chemical inhibitor experiments. White cells represent undifferentiated basal cells. Yellow and purple depict squamous and mucinous-differentiating cells, respectively. Red denotes cells that have been transduced with Lenti-SOX2. (C) Summary of PI3K chemical inhibitor data. Lentivirally infected cultures were co-treated with 2.5 μM BKM120, 4 μM LY294002, or DMSO vehicle. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. BKM120-treated p-values include *p = 0.04, **p = 0.004 (IVL), ***p = 0.0001 (TMPRSS11B), 0.0002 (SPRR1A), and 0.000002 (SPRR3). LY294002-treated p-values include **p = 0.0006 (TMPRSS11B), 0.001 (IVL), ***p = 0.0004 (SPRR1A), and 0.0002 (SPRR3). For AKT immunoblotting, lysates were prepared at 2 hr (LY294002) and 24 hr (BKM120) post-drug addition, while for SOX2 immunoblotting, at 4 d post-drug addition. (D) Schematic for the shPIK3CA experiments. Cell colors are as described in (B). (E) Summary of shPIK3CA data. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.01, **p = 0.003 (SOX2), 0.002 (SPRR1A), ***p = 0.0004 (PIK3CA), 0.0001 (TMPRSS11B), 0.00001 (IVL), 0.0002 (SPRR2A). All plotted numerical data are in S1 Data.
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pbio.1002581.g005: PI3K signaling is necessary for the squamous response to SOX2.(A) The PI3K inhibitor BKM120 inhibits growth of tracheobronchial basal cells. Basal cells were grown on plastic over 7 d with fresh media and drug added every other day. Growth was quantified by alamarBlue and is normalized to control cells treated with the DMSO vehicle, which was given a value of 100%. The means ± SEM from three replicates are shown. (B-E) Inhibition of PI3K signaling prevents SOX2-driven squamous differentiation. Tracheobronchial basal cells growing on plastic were infected with Lenti-SOX2 or empty vector, co-treated with the indicated drugs or shRNA viruses for 5 d, and then assayed for lineage marker expression by qRT-PCR. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. Because the magnitudes of inductions sometimes varied between biological replicate experiments, fold-inductions were directly compared between matched pairs of Lenti-SOX2 (with DMSO or shluc) and PI3K-inhibited (chemical inhibitor or shPIK3CA) Lenti-SOX2-transduced cultures. The amount of marker induction in inhibitor-treated cultures was then plotted as a percentage of the induction response seen without inhibitor treatment, which was given a value of 100. (B) Schematic for the PI3K chemical inhibitor experiments. White cells represent undifferentiated basal cells. Yellow and purple depict squamous and mucinous-differentiating cells, respectively. Red denotes cells that have been transduced with Lenti-SOX2. (C) Summary of PI3K chemical inhibitor data. Lentivirally infected cultures were co-treated with 2.5 μM BKM120, 4 μM LY294002, or DMSO vehicle. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. BKM120-treated p-values include *p = 0.04, **p = 0.004 (IVL), ***p = 0.0001 (TMPRSS11B), 0.0002 (SPRR1A), and 0.000002 (SPRR3). LY294002-treated p-values include **p = 0.0006 (TMPRSS11B), 0.001 (IVL), ***p = 0.0004 (SPRR1A), and 0.0002 (SPRR3). For AKT immunoblotting, lysates were prepared at 2 hr (LY294002) and 24 hr (BKM120) post-drug addition, while for SOX2 immunoblotting, at 4 d post-drug addition. (D) Schematic for the shPIK3CA experiments. Cell colors are as described in (B). (E) Summary of shPIK3CA data. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.01, **p = 0.003 (SOX2), 0.002 (SPRR1A), ***p = 0.0004 (PIK3CA), 0.0001 (TMPRSS11B), 0.00001 (IVL), 0.0002 (SPRR2A). All plotted numerical data are in S1 Data.

Mentions: To determine if PI3K signaling is necessary for the squamous response to SOX2, we treated proliferating basal cell cultures on plastic with pan-class I/II/III and class I-specific PI3K inhibitors LY294002 and BKM120, respectively [85,86], concurrently with Lenti-SOX2 infection. Due to growth suppression by these drugs (Fig 5A), assays were limited to short-term plastic cultures. Both drugs inhibited AKT activation and suppressed squamous, but not mucinous, differentiation, without affecting Lenti-SOX2 expression (Fig 5B and 5C). Notably, in some experiments, the class I (p110α, β, δ, γ)-specific drug, BKM120, enhanced MUC16 expression. We corroborated these findings with shRNA knockdown of PIK3CA, which is in the 3q amplicon. shPIK3CA reduced SOX2-dependent squamous differentiation and enhanced MUC16 expression (Fig 5E), suggesting p110α may not only promote squamous differentiation but may also dampen the mucinous response to SOX2.


SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis
PI3K signaling is necessary for the squamous response to SOX2.(A) The PI3K inhibitor BKM120 inhibits growth of tracheobronchial basal cells. Basal cells were grown on plastic over 7 d with fresh media and drug added every other day. Growth was quantified by alamarBlue and is normalized to control cells treated with the DMSO vehicle, which was given a value of 100%. The means ± SEM from three replicates are shown. (B-E) Inhibition of PI3K signaling prevents SOX2-driven squamous differentiation. Tracheobronchial basal cells growing on plastic were infected with Lenti-SOX2 or empty vector, co-treated with the indicated drugs or shRNA viruses for 5 d, and then assayed for lineage marker expression by qRT-PCR. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. Because the magnitudes of inductions sometimes varied between biological replicate experiments, fold-inductions were directly compared between matched pairs of Lenti-SOX2 (with DMSO or shluc) and PI3K-inhibited (chemical inhibitor or shPIK3CA) Lenti-SOX2-transduced cultures. The amount of marker induction in inhibitor-treated cultures was then plotted as a percentage of the induction response seen without inhibitor treatment, which was given a value of 100. (B) Schematic for the PI3K chemical inhibitor experiments. White cells represent undifferentiated basal cells. Yellow and purple depict squamous and mucinous-differentiating cells, respectively. Red denotes cells that have been transduced with Lenti-SOX2. (C) Summary of PI3K chemical inhibitor data. Lentivirally infected cultures were co-treated with 2.5 μM BKM120, 4 μM LY294002, or DMSO vehicle. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. BKM120-treated p-values include *p = 0.04, **p = 0.004 (IVL), ***p = 0.0001 (TMPRSS11B), 0.0002 (SPRR1A), and 0.000002 (SPRR3). LY294002-treated p-values include **p = 0.0006 (TMPRSS11B), 0.001 (IVL), ***p = 0.0004 (SPRR1A), and 0.0002 (SPRR3). For AKT immunoblotting, lysates were prepared at 2 hr (LY294002) and 24 hr (BKM120) post-drug addition, while for SOX2 immunoblotting, at 4 d post-drug addition. (D) Schematic for the shPIK3CA experiments. Cell colors are as described in (B). (E) Summary of shPIK3CA data. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.01, **p = 0.003 (SOX2), 0.002 (SPRR1A), ***p = 0.0004 (PIK3CA), 0.0001 (TMPRSS11B), 0.00001 (IVL), 0.0002 (SPRR2A). All plotted numerical data are in S1 Data.
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pbio.1002581.g005: PI3K signaling is necessary for the squamous response to SOX2.(A) The PI3K inhibitor BKM120 inhibits growth of tracheobronchial basal cells. Basal cells were grown on plastic over 7 d with fresh media and drug added every other day. Growth was quantified by alamarBlue and is normalized to control cells treated with the DMSO vehicle, which was given a value of 100%. The means ± SEM from three replicates are shown. (B-E) Inhibition of PI3K signaling prevents SOX2-driven squamous differentiation. Tracheobronchial basal cells growing on plastic were infected with Lenti-SOX2 or empty vector, co-treated with the indicated drugs or shRNA viruses for 5 d, and then assayed for lineage marker expression by qRT-PCR. Fold inductions were first calculated by comparing marker expression between Lenti-SOX2 and control vector (non-SOX2)-transduced cells. Because the magnitudes of inductions sometimes varied between biological replicate experiments, fold-inductions were directly compared between matched pairs of Lenti-SOX2 (with DMSO or shluc) and PI3K-inhibited (chemical inhibitor or shPIK3CA) Lenti-SOX2-transduced cultures. The amount of marker induction in inhibitor-treated cultures was then plotted as a percentage of the induction response seen without inhibitor treatment, which was given a value of 100. (B) Schematic for the PI3K chemical inhibitor experiments. White cells represent undifferentiated basal cells. Yellow and purple depict squamous and mucinous-differentiating cells, respectively. Red denotes cells that have been transduced with Lenti-SOX2. (C) Summary of PI3K chemical inhibitor data. Lentivirally infected cultures were co-treated with 2.5 μM BKM120, 4 μM LY294002, or DMSO vehicle. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. BKM120-treated p-values include *p = 0.04, **p = 0.004 (IVL), ***p = 0.0001 (TMPRSS11B), 0.0002 (SPRR1A), and 0.000002 (SPRR3). LY294002-treated p-values include **p = 0.0006 (TMPRSS11B), 0.001 (IVL), ***p = 0.0004 (SPRR1A), and 0.0002 (SPRR3). For AKT immunoblotting, lysates were prepared at 2 hr (LY294002) and 24 hr (BKM120) post-drug addition, while for SOX2 immunoblotting, at 4 d post-drug addition. (D) Schematic for the shPIK3CA experiments. Cell colors are as described in (B). (E) Summary of shPIK3CA data. Means ± SEM of three replicates are shown. Significance was calculated using paired two-tailed t tests. *p = 0.01, **p = 0.003 (SOX2), 0.002 (SPRR1A), ***p = 0.0004 (PIK3CA), 0.0001 (TMPRSS11B), 0.00001 (IVL), 0.0002 (SPRR2A). All plotted numerical data are in S1 Data.
Mentions: To determine if PI3K signaling is necessary for the squamous response to SOX2, we treated proliferating basal cell cultures on plastic with pan-class I/II/III and class I-specific PI3K inhibitors LY294002 and BKM120, respectively [85,86], concurrently with Lenti-SOX2 infection. Due to growth suppression by these drugs (Fig 5A), assays were limited to short-term plastic cultures. Both drugs inhibited AKT activation and suppressed squamous, but not mucinous, differentiation, without affecting Lenti-SOX2 expression (Fig 5B and 5C). Notably, in some experiments, the class I (p110α, β, δ, γ)-specific drug, BKM120, enhanced MUC16 expression. We corroborated these findings with shRNA knockdown of PIK3CA, which is in the 3q amplicon. shPIK3CA reduced SOX2-dependent squamous differentiation and enhanced MUC16 expression (Fig 5E), suggesting p110α may not only promote squamous differentiation but may also dampen the mucinous response to 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