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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.


PI3K signaling is high in proliferating basal cells and in squamous differentiating epithelia.(A) PIK3CA is co-amplified with SOX2 at 3q26-28 in lung SQCCs. SOX2 and PIK3CA copy number variation data for 177 primary patient SQCCs from the TCGA. Numerical data are in S1 Data. (B) PI3K activity in ALI cultures of tracheobronchial basal cells. Basal cells were infected with Lenti-SOX2 or control vector and grown at ALI, as described in Fig 2A. Cultures were stained for phospho-Ser240/244-S6 (P-S6) or phospho-Thr308-AKT (P-AKT) at the indicated times. Dotted lines outline areas of squamous metaplasia, including upper differentiated layers that had detached during sectioning. Orange arrows mark basal cells in squamous metaplasia that are stained positive for nuclear P-AKT. Scale bars are 20 μm.
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pbio.1002581.g004: PI3K signaling is high in proliferating basal cells and in squamous differentiating epithelia.(A) PIK3CA is co-amplified with SOX2 at 3q26-28 in lung SQCCs. SOX2 and PIK3CA copy number variation data for 177 primary patient SQCCs from the TCGA. Numerical data are in S1 Data. (B) PI3K activity in ALI cultures of tracheobronchial basal cells. Basal cells were infected with Lenti-SOX2 or control vector and grown at ALI, as described in Fig 2A. Cultures were stained for phospho-Ser240/244-S6 (P-S6) or phospho-Thr308-AKT (P-AKT) at the indicated times. Dotted lines outline areas of squamous metaplasia, including upper differentiated layers that had detached during sectioning. Orange arrows mark basal cells in squamous metaplasia that are stained positive for nuclear P-AKT. Scale bars are 20 μm.

Mentions: Since squamous metaplasia is not observed in ALI cultures when SOX2 levels naturally rise after quiescence but occurs if SOX2 is precociously expressed during the proliferative phase of culture, a signal associated with this phase may cooperate with SOX2 to induce the squamous response. Cigarette smoke extract and nicotine induce activation of AKT, a downstream effector of PI3K signaling, in tracheobronchial basal cell cultures [72,73], and PI3K signaling is elevated in dysplasias when SOX2 amplification is common [37,38,74–77]. Furthermore, AKT can phosphorylate SOX2 [78,79], and PIK3CA, which encodes the p110α catalytic subunit of PI3K, is in the 3q amplicon, with gains co-occurring in 99% of SQCCs with SOX2 gains (Fig 4A) [42]. To investigate the role of PI3K signaling in squamous differentiation, we first examined its activity in proliferating versus quiescing basal cell cultures. To assess PI3K signaling, we initially focused on phosphorylation of the S6 ribosomal subunit on sites regulated by a PI3K-mTOR-S6 kinase axis [80–82]. In control ALI cultures, P-S6 was high during the proliferative SOX2Lo phase but declined at initial confluence, before SOX2 levels normally rise and mucociliary differentiation occurs (Figs 4B, 1C and 1D for SOX2 expression). Nuclear accumulation of phospho-Thr308-AKT, which is PI3K-dependent [83], tracked with P-S6 and provided additional evidence that PI3K signaling is more active in proliferating rather than quiescing basal cells (Fig 4B). Consistent with the ALI data, P-S6 and nuclear P-AKT staining were also low in basal cells of quiescent native tracheal epithelia (S3 Fig). Thus, PI3K signaling is specifically active during the period when SOX2 levels are normally low and precocious SOX2 expression causes later manifestation of squamous metaplasia. Notably, by contrast to control differentiated cultures, in 5-wk Lenti-SOX2-transduced ALI cultures, PI3K signaling was active, but only in squamous metaplasias and not adjacent glandular areas (Fig 4B). This finding is consistent with premalignant squamous lesions having elevated PI3K signaling [74–77]. It also suggests that SOX2 may be able to amplify PI3K signaling in certain cell contexts such as squamous-committed cells, which has been observed in esophageal SQCC cell lines [84].


SOX2 and PI3K Cooperate to Induce and Stabilize a Squamous-Committed Stem Cell Injury State during Lung Squamous Cell Carcinoma Pathogenesis
PI3K signaling is high in proliferating basal cells and in squamous differentiating epithelia.(A) PIK3CA is co-amplified with SOX2 at 3q26-28 in lung SQCCs. SOX2 and PIK3CA copy number variation data for 177 primary patient SQCCs from the TCGA. Numerical data are in S1 Data. (B) PI3K activity in ALI cultures of tracheobronchial basal cells. Basal cells were infected with Lenti-SOX2 or control vector and grown at ALI, as described in Fig 2A. Cultures were stained for phospho-Ser240/244-S6 (P-S6) or phospho-Thr308-AKT (P-AKT) at the indicated times. Dotted lines outline areas of squamous metaplasia, including upper differentiated layers that had detached during sectioning. Orange arrows mark basal cells in squamous metaplasia that are stained positive for nuclear P-AKT. Scale bars are 20 μm.
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pbio.1002581.g004: PI3K signaling is high in proliferating basal cells and in squamous differentiating epithelia.(A) PIK3CA is co-amplified with SOX2 at 3q26-28 in lung SQCCs. SOX2 and PIK3CA copy number variation data for 177 primary patient SQCCs from the TCGA. Numerical data are in S1 Data. (B) PI3K activity in ALI cultures of tracheobronchial basal cells. Basal cells were infected with Lenti-SOX2 or control vector and grown at ALI, as described in Fig 2A. Cultures were stained for phospho-Ser240/244-S6 (P-S6) or phospho-Thr308-AKT (P-AKT) at the indicated times. Dotted lines outline areas of squamous metaplasia, including upper differentiated layers that had detached during sectioning. Orange arrows mark basal cells in squamous metaplasia that are stained positive for nuclear P-AKT. Scale bars are 20 μm.
Mentions: Since squamous metaplasia is not observed in ALI cultures when SOX2 levels naturally rise after quiescence but occurs if SOX2 is precociously expressed during the proliferative phase of culture, a signal associated with this phase may cooperate with SOX2 to induce the squamous response. Cigarette smoke extract and nicotine induce activation of AKT, a downstream effector of PI3K signaling, in tracheobronchial basal cell cultures [72,73], and PI3K signaling is elevated in dysplasias when SOX2 amplification is common [37,38,74–77]. Furthermore, AKT can phosphorylate SOX2 [78,79], and PIK3CA, which encodes the p110α catalytic subunit of PI3K, is in the 3q amplicon, with gains co-occurring in 99% of SQCCs with SOX2 gains (Fig 4A) [42]. To investigate the role of PI3K signaling in squamous differentiation, we first examined its activity in proliferating versus quiescing basal cell cultures. To assess PI3K signaling, we initially focused on phosphorylation of the S6 ribosomal subunit on sites regulated by a PI3K-mTOR-S6 kinase axis [80–82]. In control ALI cultures, P-S6 was high during the proliferative SOX2Lo phase but declined at initial confluence, before SOX2 levels normally rise and mucociliary differentiation occurs (Figs 4B, 1C and 1D for SOX2 expression). Nuclear accumulation of phospho-Thr308-AKT, which is PI3K-dependent [83], tracked with P-S6 and provided additional evidence that PI3K signaling is more active in proliferating rather than quiescing basal cells (Fig 4B). Consistent with the ALI data, P-S6 and nuclear P-AKT staining were also low in basal cells of quiescent native tracheal epithelia (S3 Fig). Thus, PI3K signaling is specifically active during the period when SOX2 levels are normally low and precocious SOX2 expression causes later manifestation of squamous metaplasia. Notably, by contrast to control differentiated cultures, in 5-wk Lenti-SOX2-transduced ALI cultures, PI3K signaling was active, but only in squamous metaplasias and not adjacent glandular areas (Fig 4B). This finding is consistent with premalignant squamous lesions having elevated PI3K signaling [74–77]. It also suggests that SOX2 may be able to amplify PI3K signaling in certain cell contexts such as squamous-committed cells, which has been observed in esophageal SQCC cell lines [84].

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.