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. 2011 May;9(5):637-47.
doi: 10.1158/1541-7786.MCR-09-0237. Epub 2011 Feb 25.

Identification of CD44 as a surface biomarker for drug resistance by surface proteome signature technology

Jason W Cain  1 , Robert S Hauptschein, Jean K Stewart, Tugba Bagci, Gary G Sahagian, Daniel G Jay
Affiliations

Identification of CD44 as a surface biomarker for drug resistance by surface proteome signature technology

Jason W Cain et al. Mol Cancer Res. 2011 May.

Abstract

We developed surface proteome signatures (SPS) for identification of new biomarkers playing a role in cancer drug resistance. SPS compares surface antigen expression of different cell lines by immunocytochemistry of a phage display antibody library directed to surface antigens of HT1080 fibrosarcoma cells. We applied SPS to compare the surface proteomes of two epithelial derived cancer cell lines, MCF7 and NCI/ADR-RES, which is drug resistant because of overexpression of the P-glycoprotein (P-gp) drug efflux pump. Surface proteomic profiling identified CD44 as an additional biomarker that distinguishes between these two cell lines. CD44 immunohistochemistry can distinguish between tumors derived from these lines and predict tumor response to doxorubicin in vivo. We further show that CD44 plays a role in drug resistance, independently of P-gp, in NCI/ADR-RES cells and increases expression of the antiapoptotic protein Bcl-xL. Our findings illustrate the utility of SPS to distinguish between cancer cell lines and their derived tumors and identify novel biomarkers involved in drug resistance.

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Figures

Fig. 1
Fig. 1. SPS can detect altered expression of controlled knockdown for PVR
A, siRNA knockdown of a known scFv (5d11) cognate antigen, PVR, in HT1080 cells. Western blot analysis shows 79% knockdown of PVR. B, confirmation of knockdown by immunofluorescent microscopy using the anti-PVR monoclonal antibody D171. C, Immunocytochemistry quantification of scFv binding in PVR siRNA treated HT1080 cells. *, significant difference (P<0.0001) compared with control by t-test.
Fig. 2
Fig. 2. Pair-wise comparison of 70 scFvs SPS panel for MCF7, HT1080 and MCF7 replicates
A, MCF7 replicate comparison of (70 scFv SPS profile). Dashed line indicates 2-fold difference in binding threshold. Three scFvs that met criteria in the original SPS screen did not repeat in a confirmatory screen. B, MCF7 vs. HT1080 SPS comparison shows numerous differences in scFv antigen binding. (circle)= scFv change in binding during initial experiment, (triangle)= indicate scFv’s demonstrating significant difference in antigen binding repeating in secondary screen and are designated as potential biomarkers. C, Immunocytochemistry for scFv 2a8 and 2f8 signals for 30 individual samples performed in triplicate of HT-1080 and MCF7 cells from three separate experiments.
Fig. 3
Fig. 3. scFv 4a1 can distinguish between cell lines in vitro
A. 70 scFv SPS comparison of MCF7 and NCI/ADR-RES yielded nine candidate biomarkers. (circle)= scFv change in binding during initial experiment, (triangle)=indicate scFv’s demonstrating significant difference in antigen binding repeating in secondary screen and are designated as potential biomarkers. B. Average normalized RFU for 4a1 shown for immunocytochemistry for 30 individual samples (each sample is the average of a triplicate of 20,000 cells per well) of MCF7 and NCI/ADR-RES cells from three separate experiments. Immunofluorescent microscopy of MCF7 and NCI/ADR-RES cells confirm relative binding differences for 4a1 between cell lines.
Fig. 4
Fig. 4. CD44 distinguishes MCF7 from NCI/ADR-RES derived tumors
A. Analysis of average tumor growth for both treated (n=15) and untreated (n=12) tumors as indicated by light emission (photon/second) upon luciferase activity. Starting at week 4, a subset of each tumor cohort (MCF7 n=8, NCI/ADR-RES n=7) were treated with doxorubicin (2mg/kg, weekly injection) B. Immunohistochemical staining of NCI/ADR-RES cells and xenograft tumors with anti-CD44 antibody (10× magnification) C. Doxorubicin untreated tumor sections were scored for CD44 staining in a blinded fashion using (−/+)-(+++) scaled scoringD. CD44 scoring of doxorubicin treated tumors. Red type indicates tumors staining inconsistent with tumor identity.
Fig 5
Fig 5. CD44 and MDR1/p-glycoprotein act in doxorubicin resistance by distinct mechanisms
A, Independent function of CD44 and p-glycoprotein was assessed by RNAi co-knockdown (Western blot) effect on doxorubicin sensitivity. Inset table, average IC50 dose for siRNA treated cells. Data reflects average values for two separate experiments. Statistical significance was established via t-test (P< 0.05) by comparing siRNA treatment to control IC50. B, NCI/ADR-RES cells depleted of CD44 and p-glycoprotein expression was confirmed via western blot and assayed for efflux of 3,3 diethyloxacarbocyanine iodide dye to determine p-glycoprotein activity.
Fig. 6
Fig. 6. Bcl-xL expression is increased in NCI/ADR-RES cells challenged with Hyaluronan
A. Bcl-xL expression was measured in serum starved MCF7, NCI/ADR-RES and CD44 siRNA NCI/ADR-RES cells treated with +/− 50 μg/ml hyaluronan polymers for 24 hours. Ratio values represent fold difference of siRNA treated lysates compared to control. β-actin expression was determined for MCF7 and NCI/ADR-RES +/− CD44 siRNA for loading control. B. Functional relevance of Bcl-xL in drug resistance was tested using NCI/ADR-RES, CD44 siRNA NCI/ADR-RES and CD44 siRNA NCI/ADR-RES cells overexpressing Bcl-xL. Cells were treated with increasing levels of Doxorubicin for 24 hours and viability was determined by CellTiter Glo assay.
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