Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide and is characterized by a fast-paced growth. Like other solid tumors, the HNSCC growth rate results in the development of hypoxic regions identified by the expression of hypoxia-inducible factor 1α (HIF-1α). Interestingly, clinical data have shown that pharmacological induction of intratumoral hypoxia caused an unexpected rise in tumor metastasis and the accumulation of cancer stem cells (CSCs). However, little is known on the molecular circuitries involved in the presence of intratumoral hypoxia and the augmented population of CSCs. Here we explore the impact of hypoxia on the behavior of HNSCC and define that the controlling function of phosphatase and tensin homolog (PTEN) over HIF-1α expression and CSC accumulation are de-regulated during hypoxic events. Our findings indicate that hypoxic niches are poised to accumulate CSCs in a molecular process driven by the loss of PTEN activity. Furthermore, our data suggest that targeted therapies aiming at the PTEN/PI3K signaling may constitute an effective strategy to counteract the development of intratumoral hypoxia and the accumulation of CSCs.-Nascimento-Filho, C. H. V., Webber, L. P., Borgato, G. B., Goloni-Bertollo, E. M., Squarize, C. H., Castilho, R. M. Hypoxic niches are endowed with a protumorigenic mechanism that supersedes the protective function of PTEN.

Keywords: EMT; ROS; cancer stem cell; epithelial-mesenchymal transition; mTOR.

Figure 1

HNSCC cell under hypoxic conditions underwent EMT phenotype and enhanced invasive behavior. A) Time course assay of 3 HNSCC cell lines growing under hypoxic conditions depict increased expression of HIF-1α compared with normoxic controls. Data represent means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. B) HNSCC cell lines under hypoxic conditions presenting spindle-shaped phenotype compared with normoxic condition. Data represent means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. C) Immunofluorescence for VIM of HNSCC cells cultured under hypoxia (24 h). Lower graphics depict the percentage of cancer cells expressing VIM under normoxia and hypoxia culture conditions. Data represent means ± sem. Scale bars, 100 μm. *P < 0.05, ***P < 0.001. D) Increased folds of tumor invasion of WSU-HN6 (2.56), WSU-HN12 (1.5), and WSU-HN13 (1.38) cell lines upon invasion assay under hypoxic conditions. E) Augmented invasion of hypoxic WSU-HN6, WSU-HN12, and WSU-HN13 cell lines compared with tumor cells invading in normoxia. Data represent means ± sem. *P < 0.05, ***P < 0.001. F) Quantification of the total number of viable tumor cells after 24 h of invasion at the upper and lower chambers (initial seeding density of 10⁴ cells). Note the higher number of WSU-HN6 tumor cells under hypoxia compared with the lower number of WSU-HN12 and WSU-HN13 cells compared with normoxic conditions. Data represent means ± sem. *P < 0.05, ****P < 0.0001. G) Schematic representation of the enhanced invasion of tumor cells during hypoxia.

Figure 2

PTEN down-regulation during hypoxia. A–C) Time course assay for PTEN expression levels in head and neck cancer cell lines cultured under hypoxic conditions and compared with normoxic controls Data represent means ± sem. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Note that all analyzed cancer cell lines present reduced levels of PTEN when cultured under hypoxia. D–F) Representative images from WSU-HN6, WSU-HN12, and WSU-HN13 cell lines cultured under the hypoxic condition for up to 18 h. Immunofluorescence staining for PTEN depicts down-regulation of the tumor suppressor starting after 6 h of hypoxia for WSU-HN6 and WSU-HN13 and after 12 h for WSU-HN12 compared with normoxic controls. pS6 staining was used as a readout for the activation of the PTEN/PI3K-signaling pathway. Arrows indicate high expression of pS6 after 12 h of hypoxia in all tested HNSCC cell lines. Scale bars, 100 μm.

Figure 3

Genetic deregulation of PTEN induces HIF-1α expression. A) Tumor sample (H&E) from a human HNSCC depicts the formation of a typical morphologic aspect of a fast-growing HNSCC displaying the development of multiple tumor islands composed of concentric squamous malignant epithelial cells. B) Normal histologic architecture of xenograft-derived squamous cell carcinoma (H&E) presenting tumor islands depicting different degrees of cellular differentiation. C–F) Immunofluorescence staining of squamous cell carcinoma xenografts displaying HIF-1α staining conjugated with Alexa 568 (C), PTEN staining conjugated with Alexa 488 (D, arrows), merge of all 3 channels (E) presenting cells positive for PTEN within the tumor mass (F, arrows), and within the hypoxic niche, and hypoxic areas (red). G) Down-regulation of protein levels of PTEN upon delivery of PTEN-shRNA. H) Representative image of HNSCC cells expressing high levels of HIF-1α detected by immunofluorescence after delivery of PTEN-shRNA. I) Quantification of the total number of HIF-1α–positive cells from tumor cells receiving PTEN-shRNA compared with control shRNA (scrambled shRNA). ****P < 0.0001. J) Schematic representation of hypoxic niches and the expression of PTEN-driven expression of HIF-1α. Scale bars, 100 μm.

Figure 4

Pharmacological down-regulation of PTEN activates HIF-1α expression and triggers an EMT phenotype on HNSCC. A) Administration of BpV(pic) efficiently down-regulated PTEN and NDRG2 after 48hrs of treatment. B) HNSCC tumor cells exposed to BpV(pic) acquire a fusiform phenotype. C) Administration of BpV(pic) triggers the accumulation of HIF-1α and pS6 as demonstrated by immunofluorescence staining. Note colocalization of HIF-1α and pS6 in tumor cells (arrow). D, E) Quantification of HIF-1α (D) and pS6 (E) immunostainings upon administration of BpV(pic). **P < 0.01, ***P < 0.001. F) Real-time PCR of HNSCC cells receiving the PTEN inhibitor BpV(pic) demonstrate up-regulation of VIM, Snail, and Nanog genes. ***P < 0.001, ****P < 0.0001. G) Tumor cells growing under hypoxic conditions also present higher gene expression levels of VIM, Snail, and Nanog, along with Twist-1. *P < 0.05, ***P < 0.001, ****P < 0.0001. H) Real-time PCR demonstrate down-regulation of NDRG2 in head and neck cell line during hypoxic conditions. ***P < 0.001. I) Schematic representation of similar effects of hypoxia and BpV(pic) on the down-regulation of PTEN, followed by the up-regulation of HIF-1α and pS6. Scale bars, 100 μm.

Figure 5

PTEN inhibition reduces ROS and enhances tumor proliferation. A) Flow cytometry for ROS and superoxide of HNSCC cells receiving 5 μM BpV(pic) or vehicle for 48 h. Note reduced levels of ROS and superoxide upon administration of BpV(pic). Data represent means ± sem. ****P < 0.0001. B–D) Detection of DNA synthesis using BrdU combined with flow cytometry demonstrate reduced apoptosis and increased proliferation of tumor cells receiving BpV(pic). Data represent means ± sem. *P < 0.05. E) Detection of DNA synthesis using BrdU combined with flow cytometry also identified a population of tumor cells undergoing G₀/G₁ upon administration of BpV(pic). F) Flow cytometry analysis identifies 2 distinct populations of tumor cells under G₀/G₁ and G₂/M. G) Schematic representation of PTEN inhibition associated with reduced ROS levels and reduced apoptosis, resulting in augmented tumor proliferation of HNSCC cells.

Figure 6

Accumulation of CSCs upon PTEN loss of function. A) Schematic representation of flow cytometry assay using HNSCC cell lines cultured under normoxia and hypoxic conditions and receiving BpV(pic). B, C) Tumor cells cultured under normoxia and hypoxia (12 h), showing increased levels of ALDH^Bright and CD44^high positive cells. Data represent means ± sem. *P < 0.05. D) Representative graphic depicting augmented ALDH^Bright- and CD44^high-positive tumor cells receiving BpV(pic) treatment and vehicle control for 24 h. E) Note a concentration-dependent accumulation of ALDH^Bright and CD44^high positive cells upon receiving BpV(pic) for 24 h. **P < 0.01. F) Tumor-derived from HNSCC xenografts demonstrates hypoxic niches within tumor mass expressing high levels of ALDH1A1 and HIF-1α. Arrows indicate cytoplasmic expression of HIF-1α and the arrowhead indicates ALDH1A1 positive tumor cells. Scale bars, 100 μm.

Figure 7

Schematic representation of our main findings indicates the presence of hypoxic niches in HNSCC tumors characterized by a reduced expression of PTEN. Low levels of PTEN leads to the accumulation of CSCs, along with the enhanced levels of tumor cells undergoing EMT. Along with increased invasive behavior, hypoxic niches are characterized by reduced levels of apoptosis resulted from diminished levels of ROS.