Expression of the Adenovirus Early Gene 1A Transcription-Repression Domain Alone Downregulates HER2 and Results in the Death of Human Breast Cancer Cells Upregulated for the HER2 Proto-Oncogene

Abstract

Adenovirus (Ad) early gene 1A 243 residue protein (E1A 243R) possesses a potent transcription-repression function within the N-terminal 80 amino acids (E1A 1-80). We examined the ability of E1A 243R and E1A 1-80 to repress transcription of both an exogenous and the endogenous HER2 promoter in a human breast cancer cell line upregulated for the HER2 proto-oncogene (SK-BR-3). Both moieties repressed HER2 expression by over 90%. When E1A 1-80 was expressed from a nonreplicative Ad vector, levels of expression were lower than anticipated. Addition of nonspecific sequences to the E1A 1-80 C-terminus (E1A 1-80 C+) enhanced its expression 10- to 20-fold. Because "oncogene addiction" suggests that repression of HER2 could kill HER2 upregulated cells, we examined the ability of full-length E1A 243R and E1A 1-80 C+ delivered by an Ad vector to kill HER2 upregulated SK-BR-3 cells. Expression of both E1A 243R and E1A 1-80 C+ killed SK-BR-3 cells but not normal breast cells. E1A 1-80 C+ is a particularly effective killer of SK-BR-3 cells. At 144 h post infection, over 85% of SK-BR-3 cells were killed by a 100 moi of the Ad vector expressing E1A 1-80 C+. As controls, Ad vectors expressing E1A 243R with deletion of all known functional domains or expressing unrelated β-galactosidase had no effect. Three additional human breast cancer cells lines reported to be upregulated for HER2 or another EGF family member (EGFR) were found to be efficiently killed by expression of E1A 1-80 C+, whereas three additional "normal" cell lines (two derived from breast and one from foreskin) were not. The ability of the E1A transcription-repression domain alone to kill HER2 upregulated breast cancer cells has potential for development of therapies for treatment of aggressive human breast cancers and potentially other human cancers that overexpress HER2.

Keywords: E1A; HER2; adenovirus; oncogene addiction; transcription repression.

Figure 1.

Transcription from an exogenous HER2 promoter is repressed by E1A 243R and E1A 1-80. (A) Normal human breast cells, MCF-10A, were transfected with a plasmid expressing the luciferase gene driven by the HER2 promoter and co-transfected with increasing amounts pDest47 expressing E1A 243R. Cells were harvested 48 h post transfection, and luciferase gene expression measured as described in the Materials and Methods section. Data are from a representative experiment. (B) Human breast cancer cells, SK-BR-3, were transfected with a plasmid expressing the luciferase gene driven by the HER2 promoter and co-transfected with pcDNA3 or with pcDNA3 expressing E1A 243R or E1A 1-80. Cells were harvested 48 h post transfection, and luciferase gene expression measured as described in the Materials and Methods. Data are from a representative experiment.

Figure 2.

E1A 243R, when expressed from an Ad vector, is able to repress the transcription of the endogenous HER2 promoter in SK-BR-3 human breast cancer cells. Cells were infected with 30 or 300 moi of AdCMV E1A 243R and harvested at 36 h PI. Expression of HER2 was measured by quantitative RT-PCR. Data are from a representative experiment.

Figure 3.

E1A 1-80 modified at its C-terminus (E1A 1-80C+) is expressed from an Ad vector at high levels. (A) A549 cells were infected at 30 or 300 moi and subjected to Western blot analysis as described in the Materials and Methods section. E1A 1-80 C+ is expressed at much higher levels than E1A 1-80. (B) Pulse-chase analysis demonstrates that the turnover rate for E1A 1-80 and E1A 1-80 C+ proteins are approximately the same. The relative amounts of expression are indicated below each lane. Levels of expression were quantitated by phosphorimage analysis using a Typhoon Trio (GE Healthcare, Piscataway, NJ). Data are from a representative experiment.

Figure 4.

(A) SK-BR-3 human breast cancer cells or (B) HS 579.Mg normal human breast cells were mock infected (•–•) or were infected with the indicated moi of AdCMV E1A 243R (■–■), AdCMV E1A 1-80 C+ (*–*), AdCMV E1A 243R dl1101/1108/1135 (▲–▲) or AdCMV LacZ (◆–◆). Cell viability was measured as described in the Materials and Methods section. Results are the average of three or more experiments.

Figure 5.

Human breast cancer cells, MB-468 (■–■), MB-453 (◆–◆) and MB-231 (▲–▲) or normal human cells, MCF 10A (•–•), MCF 12F (X–X) and HS 68 (*–*) were infected with 300 moi of AdCMV E1A 1-80 C+ and cell viability measured as described in the Materials and Methods. Results are the average of three or more experiments except for HS 68, which is the average of two experiments.