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Comparative Study
. 2016 Jul 13:16:471.
doi: 10.1186/s12885-016-2509-5.

SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer

Ida V Lundberg  1 , Sofia Edin  2 , Vincy Eklöf  1 , Åke Öberg  3 , Richard Palmqvist  1 , Maria L Wikberg  1
Affiliations
Comparative Study

SOX2 expression is associated with a cancer stem cell state and down-regulation of CDX2 in colorectal cancer

Ida V Lundberg et al. BMC Cancer. .

Abstract

Background: To improve current treatment strategies for patients with aggressive colorectal cancer (CRC), the molecular understanding of subgroups of CRC with poor prognosis is of vast importance. SOX2 positive tumors have been associated with a poor patient outcome, but the functional role of SOX2 in CRC patient prognosis is still unclear.

Methods: An in vitro cell culture model expressing SOX2 was used to investigate the functional role of SOX2 in CRC. In vitro findings were verified using RNA from fresh frozen tumor tissue or immunohistochemistry on formalin fixed paraffin embedded (FFPE) tumor tissue from a cohort of 445 CRC patients.

Results: Using our in vitro model, we found that SOX2 expressing cells displayed several characteristics of cancer stem cells; such as a decreased proliferative rate, a spheroid growth pattern, and increased expression of stem cell markers CD24 and CD44. Cells expressing SOX2 also showed down-regulated expression of the intestinal epithelial marker CDX2. We next evaluated CDX2 expression in our patient cohort. CDX2 down-regulation was more often found in right sided tumors of high grade and high stage. Furthermore, a decreased expression of CDX2 was closely linked to MSI, CIMP-high as well as BRAF mutated tumors. A decreased expression of CDX2 was also, in a stepwise manner, strongly correlated to a poor patient prognosis. When looking at SOX2 expression in relation to CDX2, we found that SOX2 expressing tumors more often displayed a down-regulated expression of CDX2. In addition, SOX2 expressing tumors with a down-regulated CDX2 expression had a worse patient prognosis compared to those with retained CDX2 expression.

Conclusions: Our results indicate that SOX2 expression induces a cellular stem cell state in human CRC with a decreased expression of CDX2. Furthermore, a down-regulated expression of CDX2 results in a poor patient prognosis in CRC and at least part of the prognostic importance of SOX2 is mediated through CDX2 down-regulation.

Keywords: CDX2; Cancer stem cell; Colorectal cancer; Prognosis; SOX2.

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Figures

Fig. 1
Fig. 1
Evaluation of EMT and cellular migration in response to SOX2 expression. Caco2 cells and Caco2 cells stably transfected with SOX2 (Caco2-SOX2) was analyzed by RT-PCR for the expression of a EMT related genes, or b MMPs. Shown is relative gene expression from three or more independent experiments ± SD with Caco2 levels set as 1. c Cellular migration was evaluated using Boyden chamber experiments. Shown is mean number of migrating cells ± SD from three independent experiments. Significant differences are indicated by * (P < 0.05)
Fig. 2
Fig. 2
SOX2 induces a CSC state in CRC cells. Factors associated with a CSC state was evaluated in Caco2 cells and Caco2 cells stably transfected with SOX2 (Caco2-SOX2). a Proliferation of cells as measured by XTT cell proliferation assay. b Morphological evaluation of cells. c Expression of cellular adhesion molecules as evaluated by RT-PCR. Shown is relative expression with Caco2 cells set as 1. d Expression of cancer stem cell markers as evaluated by RT-PCR. Shown is relative expression with Caco2 cells set as 1. Gene expression analyses were reproduced three times and mean values ± SD is shown. Significant differences are indicated by * (P < 0.05)
Fig. 3
Fig. 3
SOX2 is associated with down-regulated expression of CDX2. a Caco2, Caco2-SOX2, SW480 and SW620 cells were analyzed by RT-PCR for the expression of CDX2 and SOX2. Shown is relative gene expression from three independent experiments ± SD with Caco2 or SW480 levels set as 1. Significant differences are indicated by * (P < 0.05). b RNA from fresh frozen tumor tissue from 25 CRC patients was analyzed by RT-PCR for the expression of SOX2 and CDX2. Shown is normalized gene expression
Fig. 4
Fig. 4
Evaluation of CDX2 expression in CRC. a Representative images of immunohistological stainings of CDX2 in human CRC tissue specimens; normal colon epithelium and CRC with <5 % expression, 5–50 % expression and >50 % expression of CDX2. b Kaplan-Meier survival analyses of CDX2 expression in CRC. c Kaplan-Meier survival analyses in subgroups of CRC defined as SOX2 positive or negative, and CDX2 < 50 % expression and >50 % expression. Log-rank tests were used to calculate P-values
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