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. 2016 Jan 19;113(3):638-43.
doi: 10.1073/pnas.1514663113. Epub 2016 Jan 6.

SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis

Sait Ozturk  1 , Panagiotis Papageorgis  2 , Chen Khuan Wong  2 , Arthur W Lambert  3 , Hamid M Abdolmaleky  4 , Arunthathi Thiagalingam  4 , Herbert T Cohen  5 , Sam Thiagalingam  6
Affiliations

SDPR functions as a metastasis suppressor in breast cancer by promoting apoptosis

Sait Ozturk et al. Proc Natl Acad Sci U S A. .

Abstract

Metastatic dissemination of breast cancer cells represents a significant clinical obstacle to curative therapy. The loss of function of metastasis suppressor genes is a major rate-limiting step in breast cancer progression that prevents the formation of new colonies at distal sites. However, the discovery of new metastasis suppressor genes in breast cancer using genomic efforts has been slow, potentially due to their primary regulation by epigenetic mechanisms. Here, we report the use of model cell lines with the same genetic lineage for the identification of a novel metastasis suppressor gene, serum deprivation response (SDPR), localized to 2q32-33, a region reported to be associated with significant loss of heterozygosity in breast cancer. In silico metaanalysis of publicly available gene expression datasets suggests that the loss of expression of SDPR correlates with significantly reduced distant-metastasis-free and relapse-free survival of breast cancer patients who underwent therapy. Furthermore, we found that stable SDPR overexpression in highly metastatic breast cancer model cell lines inhibited prosurvival pathways, shifted the balance of Bcl-2 family proteins in favor of apoptosis, and decreased migration and intravasation/extravasation potential, with a corresponding drastic suppression of metastatic nodule formation in the lungs of NOD/SCID mice. Moreover, SDPR expression is silenced by promoter DNA methylation, and as such it exemplifies epigenetic regulation of metastatic breast cancer progression. These observations highlight SDPR as a potential prognostic biomarker and a target for future therapeutic applications.

Keywords: SDPR; breast cancer; epigenetics; metastasis; metastasis suppressor.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Identification of SDPR as a candidate metastasis suppressor gene. (A) Schematic depiction of the generation of breast cancer progression cell line model system. The model consists of five cell lines representing different stages of breast cancer progression. MI, normal breast epithelial cells; NeoT and MII, carcinoma in situ; MIII, carcinoma; and MIV, metastatic carcinoma. (B) Hierarchical clustering of gene expression profiles from MII, MIII, and MIV cells for the genes whose expression differ at least twofold between each cell line. Two clusters, cluster 6 and 7, are magnified because expressions of the genes in these two clusters are significantly suppressed in metastatic MIV cells compared with nonmetastatic MII and MIII. (C) The schematic summary of our strategy for the selection of SDPR as the top candidate metastasis suppressor.
Fig. 2.
Fig. 2.
Expression analysis of SDPR in clinical samples and model cell lines. (A) SDPR mRNA levels in metastatic MIV cells compared with nonmetastatic MII (P = 0.00047) and MIII (P = 0.0005) cells. (B) Endogenous SDPR protein levels in the model cell lines were assessed by Western blot. (C) In silico Kaplan–Meier analysis depicting the association between SDPR expression and distant-metastasis–free survival (DMFS). The analysis was run on a cohort with 1,211 breast cancer patients, P = 0.0086. (D) In silico Kaplan–Meier analysis depicting the association between SDPR expression and relapse-free survival (RFS). The analysis was run on a cohort with 2,785 breast cancer patients, P = 1.1e-10. *P < 0.05.
Fig. 3.
Fig. 3.
SDPR suppresses lung colonization of breast cancer. (A) Bioluminescent imaging of animals 77 d after tail vein injections with 5 × 105 control, MIVpQ, or MIVpQ.SDPR cells. (B) The percentage of animals that developed lung metastases following tail vein injections is shown. (C) Quantification of metastases burden on mice was estimated by photon flux measurement, P = 0.026. (D) The average number of lung macrometastases observed per animal, P = 0.012. (E) SDPR overexpression was assessed by Western blotting. *P < 0.05.
Fig. 4.
Fig. 4.
SDPR primes MIV cells for apoptosis and inhibits extravasation. (A) Transendothelial cell migration potential of the control and MIVpQ.SDPR cells toward serum-free or complete media was assessed 48 h after the seeding by calcein staining, P = 0.0374. RFU, relative florescence unit. (B) Effect of SDPR on the extravasation potential of LM2 cells was quantified, P = 7.87479E-07. (C) Growth of control and MIVpQ.SDPR cells were monitored over time in 3D cell culture and quantified, on the Right, by measuring colony area, P = 0.01. (D) Effect of SDPR overexpression on survivability of MIV cells was monitored 72 h after the tail vein injections by Caliper IVIS Spectrum. Whole-animal imaging is presented on the Upper Left, and extracted lungs are shown on Lower Left. Quantification of cell survivability was assessed on the Right, based on photon flux, P = 0.0014, npQ = 5, npQSDPR = 8. (E) Annexin V and PI staining were used to assess the basal level of apoptosis in control and MIVpQ.SDPR cells. Quantification of three independent Annexin V experiments is shown, P = 0.04. *P < 0.05.
Fig. 5.
Fig. 5.
SDPR function and apoptosis. (A) The effect of SDPR overexpression on the proapoptotic PUMA and Bax expression in MIV cells was evaluated by Western blotting and luciferase reporter assays, pPUMA = 0.000003, pBax = 0.03 for all n = 3. (B) Protein levels of proapoptotic Bcl2 family members, Bad, Bid, and Bim, and antiapoptotic Bcl-xL were measured by Western blotting in control and MIpQ.SDPR cells. (C) The effect of SDPR overexpression on the activity of ERK and NF-κB pathways was evaluated by Western blotting against phosphorylated Erk and p65 protein levels, respectively. (D) Total and cleaved caspase-3 protein levels in control and MIVpQ.SDPR cells were measured by Western blotting. (E) Co-IP was carried out in MIV and LM2 cells using HIS (mouse) antibody to precipitate SDPR and mouse IgG as control. Western blot was used to assess the levels of precipitated SDPR and coprecipitated Erk. (F) SDPR overexpression, directly or indirectly, causes increases in proapoptotic BCL-2 family proteins. Additionally, the levels of the antiapoptotic protein Bcl-xL and the activity of prosurvival Erk and NF-κB signaling pathways were decreased due to SDPR overexpression. Overall, SDPR can alter the balance of regulatory proteins in the apoptotic pathway to favor cell death. *P < 0.05.
Fig. 6.
Fig. 6.
Epigenetic regulation of SDPR expression. (A) The relative expression levels of SDPR in NeoT and MIV cells was measured by quantitative RT-PCR, P = 0.0127, n = 3. (B) Effect of 5-Aza treatment on SDPR mRNA levels in MIV cells, P = 0.02, n = 3. (C) Analysis of −1000 to +1000 region of SDPR transcription start site for CpG sites using the MethPrimer online tool. (D) Methylation-specific quantitative PCR was used to assess the percentage of DNA methylation at the SDPR promoter region in NeoT and MIV cell lines, P = 0.0116, n = 3. SDPR protein levels are depicted below the graph. (E) The percentage of DNA methylation at the SDPR promoter region across nonmetastatic and metastatic breast cancer cell lines, P = 0.0007. SDPR protein levels are depicted below the graph. *P < 0.05.
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