Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors.
OBJECTIVE
In a previous gene expression array study, we identified some 300 genes that were differentially expressed in human epidermal growth factor receptor tyrosine kinase 2 (HER2)-positive versus HER2-negative breast cancer cells. We have now done validation experiments on a group of three cell membrane components that had previously not been implicated in breast cancer. We also studied the expression of three other cell membrane proteins known to play a role in mammary neoplasia.


METHODS
By immunohistochemistry, we examined up to 130 archival breast carcinomas for Celsr2, E-cadherin, Kai1, and CD9 expression. The expression levels of NET-6 and TROP-2 were determined by quantitative reverse transcription-PCR in a subset of frozen tumors. We also studied fresh pellets and paraffin-embedded cell buttons of nine human breast cell lines. The relationship between the expression of all six membrane proteins and a variety of pathologic and biological variables, including estrogen receptor, HER2, and epidermal growth factor receptor status, was also examined. The NET-6 gene was transfected into a low-expressing cell line, and the effect on cellular morphology, growth, and invasion in vitro was recorded.


RESULTS
Celsr2 was down-regulated in one cell line and in 7% of breast cancers. E-cadherin, Kai1, and CD9 were down-regulated in 35%, 76%, and 79% of tumors, respectively, confirming the important role of these markers in human mammary neoplasia. In breast cancer cell lines and tissues, TROP-2 was generally expressed at low levels, although a few specimens showed relative overexpression. NET-6 levels were lower in HER2-negative breast carcinoma cells. In addition, NET-6 was markedly down-regulated in estrogen receptor-negative breast cancers, and expression was lowest in "basal-like" tumors. Ectopic expression of NET-6 in low-expressing MDA-MB-231 cells altered cellular morphology, inhibited growth in vitro, and decreased invasion in a Boyden chamber assay.


CONCLUSIONS
We have confirmed the expression of three new membrane markers that had previously not been implicated in human breast cancer, and one of them (NET-6) was correlated with HER2 and estrogen receptor status. NET-6 levels were decreased in estrogen receptor-negative and high-grade tumors, and ectopic expression of this gene had an inhibitory effect on proliferation and invasion. Thus, NET-6 may represent a novel breast cancer suppressor gene.
Huang
Huayi
H
Department of Pathology, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Groth
Jeff
J
Sossey-Alaoui
Khalid
K
Hawthorn
Lesleyann
L
Beall
Stephanie
S
Geradts
Joseph
J
D015703:Antigens, CD / Q000032:analysis; D000951:Antigens, Neoplasm / Q000235:genetics; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000235:genetics; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D015820:Cadherins / Q000032:analysis; D015815:Cell Adhesion Molecules / Q000235:genetics; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D015536:Down-Regulation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007150:Immunohistochemistry; D051861:Kangai-1 Protein; D008562:Membrane Glycoproteins / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis* / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000032:analysis; D011960:Receptors, Estrogen / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D060245:Tetraspanin 29; D059470:Tetraspanins
D015703:Antigens, CD; D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D015820:Cadherins; D015815:Cell Adhesion Molecules; D051861:Kangai-1 Protein; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D018719:Erb-b2 Receptor Tyrosine Kinases; D011960:Receptors, Estrogen; D060245:Tetraspanin 29; D059470:Tetraspanins; C497688:CD82 protein, human; C554869:CD9 protein, human; C498477:CELSR2 protein, human; C499380:TACSTD2 protein, human; C502874:TSPAN13 protein, human