The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-␣ converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by ␥-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.

Cleavable isoforms of the ErbB4 receptor tyrosine kinase release a soluble intracellular domain (ICD) that may translocate to the nucleus and regulate signaling. However, ErbB4 gene is alternatively spliced generating CYT-1 and CYT-2 isoforms with different cytoplasmic tails. Here, we addressed whether the two alternative ErbB4 ICDs of either CYT-1 (ICD1) or CYT-2 (ICD2) type differ in signaling to the nucleus. Confocal microscopy and extraction of nuclear cell fractions indicated that significantly more ICD2 translocated to the nuclei when compared to ICD1. Unlike the membrane-anchored 80 kDa fragments derived from full-length ErbB4 isoforms, the two ICDs did not differ from each other in metabolic stability or ubiquitylation. However, ICD2 was phosphorylated at tyrosine residues to a higher extent and demonstrated greater in vitro kinase activity than ICD1. Mutating the ATP-binding site within ICD2 kinase domain (ICD2 K751R) blocked its tyrosine phosphorylation and significantly reduced its nuclear translocation. When expressed in the context of full-length ErbB4, ICD2 was also more efficient than ICD1 in promoting transcriptional activation of the STAT5 target gene b-casein. These findings indicate that the two alternative ICDs of ErbB4 differ in their nuclear accumulation, and that the mechanism involves differential kinase activity but not ubiquitin-regulated ICD stability.

Loss of heterozygosity (LOH) at the distal half of chromosome arm 11q is frequent in a variety of human tumors, including breast cancer, and is often associated with poor prognosis. In an ongoing attempt to locate and characterize the main target genes within this chromosome region, we first looked for aberrations in known genes either suggested to be involved in tumorigenesis or shown to suppress tumor formation. We examined 31 primary breast tumors showing LOH in 11q21-24 for mutations in the MRE11A, CHK1, PPP2R1B, and TSLC1 genes. The absence of intragenic alterations related to cancer led us next to evaluate possible gene silencing resulting from promoter region CpG hypermethylation, using the bisulfite sequencing technique. In addition to the four genes mentioned above, we also analyzed the ATM gene, which had been investigated for certain germline mutations in an earlier study. Only the TSLC1 promoter region exhibited aberrant methylation patterns, and altogether 33% (10/30) of the successfully analyzed tumors showed evidence of elevated levels of TSLC1 CpG methylation. Ten percent (3/30) of the tumors showed significantly increased methylation. Thus, as has been shown in lung and some other forms of cancer, hypermethylation of the TSLC1 promoter region is also frequently a second hit along with LOH in breast cancer.