Cell Specific CD44 Expression in Breast Cancer Requires the Interaction of AP-1 and NFκB with a Novel <em>cis</em>-Element

<div><p>Breast cancers contain a heterogeneous population of cells with a small percentage that possess properties similar to those found in stem cells. One of the widely accepted markers of breast cancer stem cells (BCSCs) is the cell surface marker CD44. As a glycoprotein, CD44 is involved in many cellular processes including cell adhesion, migration and proliferation, making it pro-oncogenic by nature. CD44 expression is highly up-regulated in BCSCs, and has been implicated in tumorigenesis and metastasis. However, the genetic mechanism that leads to a high level of CD44 expression in breast cancer cells and BCSCs is not well understood. Here, we identify a novel <em>cis</em>-element of the CD44 directs gene expression in breast cancer cells in a cell type specific manner. We have further identified key <em>trans-acting</em> factor binding sites and nuclear factors AP-1 and NFκB that are involved in the regulation of cell-specific CD44 expression. These findings provide new insight into the complex regulatory mechanism of CD44 expression, which may help identify more effective therapeutic targets against the breast cancer stem cells and metastatic tumors.</p> </div

Expression of key factors in 3 breast cancer cell lines.

<p>Expression of key factors in 3 breast cancer cell lines.</p

CR1 directs reporter GFP expression in breast cancer cell lines.

<p>Conserved region was tested for the ability to direct reporter gene expression by transfecting breast cancer cell lines with CD44CR1-βGP-GFP construct (CD44CR1-GFP). Nuclei were stained with Hoechst 33342. (<b>A–C</b>) GFP expression in all three cell lines resulted from transfection of a positive control construct (CAG-GFP). (<b>D–F</b>) No GFP expression was detected from transfection of a negative control construct with a conserved region from NeuroD1gene. GFP expression from CR1 can be seen in MDA-MB-231 and SUM159 cells (<b>G–H</b>). However, no expression is seen in MCF7 cells (<b>I</b>).</p

Prediction of <i>cis</i>-regulatory elements for CD44 expression using sequence alignment analysis.

<p>(<b>A</b>) A genomic map of human CD44 and surrounding genes located on chromosome 11p13. (<b>B</b>) Multiple sequence alignment of homologous CD44 sequences using human sequence as baseline. 14 evolutionarily conserved regions were identified and predicted as potential <i>cis</i>-regulatory elements for CD44 expression. Conserved regions 1–3 (CR1–3) have the highest levels of conservation. Blue regions represent CD44 coding sequence. Pink regions represent non-coding sequence. Peaks surrounded by red bars are highly conserved regions that have at least 70% conservation among species. (<b>C</b>) Plasmid reporter construct containing a conserved region of CD44, a minimal beta-globin-promoter (βGP), and green fluorescent protein (GFP).</p

Specific protein factors bind with CR1.

<p>EMSAs were performed to determine the <i>in vitro</i> binding activities of nuclear protein factors with CD44CR1. (<b>A</b>) DNA probe design using conserved mouse sequence and TFBSs within each probe. Probe 1 identified binding (indicated by arrow head) in two cell lines (MDA-MB-231 and MCF7), but not observed in SUM159. (<b>B</b>) Probe 2 showed strong binding present in all three cell lines (arrowheads). (<b>C</b>) Probe 3 showed multiple shifted bands and was successfully competed away in all three cell lines using unlabeled probes. (<b>E</b>) Probe AP-1-1 showed no band shift in any of the three cell lines. (<b>F</b>) Probe AP-1-2 resulted in a band shift in all three cell lines. All band shifts were competed away with an unlabeled probe. Arrowheads indicate bands specific to MDA-MB-231 and MCF7. (<b>G</b>) Probe NFκB showed a band shift that was successfully competed away in all three cell lines.</p

AP-1-JUNB knockdown decreases CD44 expression.

<p>Sum159 cells were transfected with control and JUNB shRNA constructs and then stained for JUNB and CD44 expression. Transfection with the control, scrambled DNA shRNA construct (<b>A–E</b>) showed no change in JUNB expression (<b>B</b>, circle) or CD44 expression (<b>C</b>, circle) when compared to un-transfected cells (arrows). Transfection with the JUNB shRNA construct (<b>F–J</b>) showed a reduction in JUNB expression (<b>G</b>, circle) and CD44 expression (<b>H</b>, circle) when compared to un-transfected cells (<b>F–G</b>, arrow).</p

Differential AP-1 factor binding to CR1 in breast cancer cells.

<p>ChIP with AP-1 antibodies resulted in amplification of a region of CR1 with inverted repeat AP-1 binding sites. Rabbit IgG and anti-GFP antibody served as negative control. Representative results of at least two independent immunoprecipitation experiments and multiple independent PCR analyses are shown. Strong PCR amplification of CR1 region with JUNB binding was seen in SUM159 cells and with JUND binding in MCF7 cells.</p

Conserved transcription factor binding sites in CR1 between mouse and human.

<p>Conserved transcription factor binding sites in CR1 between mouse and human.</p

Mutation of AP-1 and NFκB binding sites in CR1 reduces reporter GFP expression.

<p>Assays using site directed mutagenesis of AP-1 and NFκB binding sites. (<b>A–I</b>) Schematic of each mutation of CR1 construct. Mutated sites are identified by a red X. (<b>A’–I’</b>) Transfection of each the constructs in SUM159 cells. (<b>J</b>) Quantification of the number of GFP-expressing cells/total number of cells counted. Control mutation at a non-conserved site (<b>B’</b>) showed no difference in GFP expression when compared to CR1 (<b>A’</b>). Single site mutations of AP-1-1, AP-1-2 and NFκB (<b>C’-E’</b>) showed a significant reduction of GFP expression compared to CR1. However, GFP expression was not eliminated entirely. Mutation of a combination of AP-1 and NFκB binding sites (<b>F’-H’</b>) did not reduce further GFP expression, however, the percentage of GFP expression was still significantly reduced compared to CR1. Mutation of all three TFBSs (<b>I’</b>) showed the greatest reduction of GFP expression. **p = < 0.0005 ***p = <1.0×10⁻⁵ (student’s t-test). Scale bar = 50 µM.</p

NFκB-p50 knockdown decreases CD44 expression.

<p>Sum159 cells were transfected with control and NFκB-p50 shRNA constructs and then stained for NFκB-p50 and CD44 expression. Transfection with the control, scrambled DNA shRNA construct (<b>A–E</b>) showed no change in NFκB-p50 expression (<b>B</b>, circle) or CD44 expression (<b>C</b>, circle) when compared to un-transfected cells (arrows). Transfection with the NFκB-p50 shRNA construct (<b>F–J</b>) showed a reduction in NFκB-p50 expression (<b>G</b>, circle) and CD44 expression (<b>H</b>, circle) when compared to un-transfected cells (<b>F–G</b>, arrow).</p