PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer - PubMed (original) (raw)

. 2008 Aug 7;27(34):4657-65.

doi: 10.1038/onc.2008.101. Epub 2008 Apr 14.

S Koujak, S Nagase, C-M Li, T Su, X Wang, M Keniry, L Memeo, A Rojtman, M Mansukhani, H Hibshoosh, B Tycko, R Parsons

Affiliations

PCDH8, the human homolog of PAPC, is a candidate tumor suppressor of breast cancer

J S Yu et al. Oncogene. 2008.

Abstract

Carcinoma is an altered state of tissue differentiation in which epithelial cells no longer respond to cues that keep them in their proper position. A break down in these cues has disastrous consequences not only in cancer but also in embryonic development when cells of various lineages must organize into discrete entities to form a body plan. Paraxial protocadherin (PAPC) is an adhesion protein with six cadherin repeats that organizes the formation and polarity of developing cellular structures in frog, fish and mouse embryos. Here we show that protocadherin-8 (PCDH8), the human ortholog of PAPC, is inactivated through either mutation or epigenetic silencing in a high fraction of breast carcinomas. Loss of PCDH8 expression is associated with loss of heterozygosity, partial promoter methylation, and increased proliferation. Complementation of mutant tumor cell line HCC2218 with wild-type PCDH8 inhibited its growth. Two tumor mutants, E146K and R343H, were defective for inhibition of cell growth and migration. Surprisingly, the E146K mutant transformed the human mammary epithelial cell line MCF10A and sustained the expression of cyclin D1 and MYC without epidermal growth factor. We propose that loss of PCDH8 promotes oncogenesis in epithelial human cancers by disrupting cell-cell communication dedicated to tissue organization and repression of mitogenic signaling.

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Figures

Figure 1

Figure 1

Homozygous deletion in a breast tumor cell line and expression of PCDH8 in normal breast and breast tumors. (a) A homozygous deletion of 13q14–21 in HCC1395 was found by RDA analysis and corroborated by SNP chip and PCR analysis. Genes that are deleted include CHM1, PCDH8, GW112, PCDH17, DIAPH3 and TDRD3. A short stretch of DNA between PCDH8 and GW112 was retained. Other homozygous deletions in this vicinity do not appear to affect PCDH8 or any other gene expressed in breast tissue (Cox et al., 2005). (b) Southern analysis confirming deletion of 13q21 in HCC1395 tumor (T) DNA but not corresponding normal (N) DNA. The blot was stripped and hybridized with a chromosome X probe to demonstrate equal loading. Arrows denote deleted DNA. (c) Reverse transcription (RT)–PCR and western blot analysis reveals PCDH8 expression in a control cell line (MCF7) and the immortalized lines M2E6E7, M3E6E7, and MCF10A (10A), and loss of expression of PCDH8 in HCC1395 (1395). (d) PCDH8 mRNA is expressed in murine hippocampus and breast duct by in situ hybridization. Hematoxylin and eosin stain of breast duct (× 1000). (e) PCDH8 expression is lost in multiple breast cancer cell lines, HCC1395, ZR75-30 (75–30), MDA-MB-435 s (435 s) and MDA-MB-436 (436), by RT–PCR and western blot.

Figure 2

Figure 2

Downregulation of PCDH8 in breast tumors. (a) Loss of heterozygosity (LOH) at markers D13S1305, D13S155 and D13S1228 is found in tumor 68T. Heterozygosity is seen in the corresponding normal tissue, 68N. A 50% or greater reduction in peak intensity was scored as a loss. The position of markers relative to the PCDH8 locus is mapped. (b) Inactivation of PCDH8 by somatic mutation in two tumors: one missense mutation G436A:E146K is found in the extracellular domain of PCDH8 in tumor 68T, and another missense mutation, G1028A:R343H, in breast cancer cell line HCC1599. (c) Southern analysis of methylation of the PCDH8 promoter. DNA was digested with one or more restriction enzymes and electrophoresis performed in lanes 1–4, where lane 1 corresponds to digestion with RsaI, lane 2 to RsaI and CfoI, lane 3 to RsaI and HpaII and lane 4 to RsaI and MspI. CfoI and HpaII are methylation sensitive enzymes; MspI is the methylation insensitive isoschizomer of HpaII. Methylation is detected in the breast cancer cell lines ZR-75-30 (75-30) and MDA-MB-435s (MDA-435s), and breast tumors 21T, 95T and 584T. Normal breast samples and tumor 33T lack methylation of PCDH8. An ANKRD3 control blot shows completion of digestion and serves as a loading control. (d) Restriction map of PCDH8 promoter. RsaI sites are denoted by tall vertical lines labeled ‘R’. CfoI, HpaII and MspI sites containing CpGs are denoted by short vertical lines. Site of probe for Southern blotting is indicated by horizontal line. (e) PCDH8 is reactivated in MDA-MB-435s treated with 5-aza-deoxycytidine (5AdC) but not PBS control. (f) Loss of expression of PCDH8 in tumors 21T and 95T correlates with promoter methylation, as shown in (c). Adjacent normal breast lobules and ducts exhibit membranous and cytoplasmic staining of PCDH8 in breast epithelial cells (× 400). (g) PCDH8 is downregulated in breast cancer cells relative to adjacent normal breast duct cells in a breast tumor biopsy (× 100). Arrowhead = normal cells. Arrow = tumor cells.

Figure 3

Figure 3

Retroviral expression of wild-type PCDH8 suppresses growth of a mutant breast cell line HCC2218. (a) Stable pools of HCC2218 express two species of PCDH8 expressed via the pBABE-puro retroviral vectors containing wild type and mutant forms of PCDH8 as detected by immunoblot. Tubulin is used as a loading control. (b) After plating 50 000 cells, cells were resuspended and counted on the indicated days. Wild-type PCDH8 reduced the number of cells relative to empty vector control. The mutant expressing cells (PCDH8K and PCDH8H) had an intermediate effect.

Figure 4

Figure 4

Wild-type, but not mutant, PCDH8 inhibits migration of normal breast epithelial cells. (a) Protein expression of PCDH8 (10A-PCDH8), PCDH8K (10A-PCDH8K) and PCDH8H (10A-PCDH8H) in MCF10A detected by immunoblot. (b) Subcellular localization of PCDH8 and PCDH8K were determined by immunofluorescence using anti-MYC 9E10 antibodies. While PCDH8 localizes at cell processes and cell–cell junctions, PCDH8K localizes to the cytoplasm and is concentrated in perinuclear regions. Corresponding 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain of PCDH8 and PCDH8K transfected cells (× 400). (c) PCDH8 expression inhibits migration. Wound healing assay reveals reduced migration of 10A-PCDH8 relative to empty vector control and 10A-PCDH8 mutant cells (× 100). At 24 h 10A-PCDH8 cells continue to have an open wound, while control and mutant cells have already repaired the wound.

Figure 5

Figure 5

PCDH8K transforms normal breast epithelial cells and increases expression of MYC and cyclin D1 in the absence of serum. (a) PCDH8K (E146K) transforms MCF10A in 2-dimensional culture on plastic (left column, × 40) and 3-dimensional culture in Matrigel (center column, × 40). PCDH8K accelerates acinus size relative to control cells, 10A-PCDH8, and 10A-PCDH8H (right column, × 400). (b, c) Quantification of aberrant 10A-PCDH8K foci. (b) When cultured on plastic, all foci visible to the naked eye were counted in 75 cm2 flasks. (c) In reduced growth factor Matrigel, spiculated acini were counted and quantified as a ratio of spiculated acini to the number of cells originally suspended in matrigel (spiculated acini per 5000 suspended cells). (d) MCF10A derivatives were grown in the absence of epidermal growth factor (EGF) and in low serum for up to 48 h and cell lysates harvested at the indicated time points. Cyclin D1 and MYC proteins expression measured by immunoblot persists in 10A-PCDH8K (10A-P8K) and 10A-RasV12 after withdrawal of EGF.

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