In Vivo and In Vitro Studies Suggest a Possible Involvement of HPV Infection in the Early Stage of Breast Carcinogenesis via APOBEC3B Induction

High prevalence of infection with high-risk human papilloma virus (HPV) ranging from 25 to 100% (average 31%) was observed in breast cancer (BC) patients in Singapore using novel DNA chip technology. Early stage of BC demonstrated higher HPV positivity, and BC positive for estrogen receptor (ER) showed significantly higher HPV infection rate. This unique association of HPV with BC in vivo prompted us to investigate a possible involvement of HPV in early stages of breast carcinogenesis. Using normal breast epithelial cells stably transfected with HPV-18, we showed apparent upregulation of mRNA for the cytidine deaminase, APOBEC3B (A3B) which is reported to be a source of mutations in BC. HPV-induced A3B overexpression caused significant γH2AX focus formation, and DNA breaks which were cancelled by shRNA to HPV18 E6, E7 and A3B. These results strongly suggest an active involvement of HPV in the early stage of BC carcinogenesis via A3B induction

A3B activation by HPV infection increases genome instability.

<p>(a) γH2AX immunofluorescence. Cells were fixed by 4% paraformaldehyde followed by staining of γH2AX protein and nuclear using anti- γH2AX Ab and DAPI respectively. (b) γH2AX western blot. Cells were lysed and subjected to western blot. The γH2AX and β-actin were detected using anti- γH2AX and β-actin Ab. (c) Comet assay. Cells were seeded onto agarose-coated slide glass after mixing with 1.5% agarose followed by lysis. Slides were subjected to electrophoresis and then genomic DNA was stained by PI. (d) Statistical analysis for panel C. Student’s t-test was performed, with <i>p</i>-values indicated on the graph.</p

Correlation of A3B expression level and HPV infection in BC patients.

<p>Total RNA from BC patients were obtained from NUHS TR. Samples were subjected to quantitative RT-PCR (qRT-PCR). The data of A3B (a) and A3G (b) were shown after normalization by GAPDH mRNA level. The number at the bottom of graph indicates the number of patients. Patient 1–11 are HPV-negative, patient 12–23 are HPV-positive.</p

Relationship between HPV status and patholological features.

<p>Numbers at the bottom of each bar represent (number of HPV-positives)/(total sample numbers). Difference of HPV prevalence between invasive ductal carcinoma (IDC) and invasive LC (ILC) was statistically significant (<i>p</i> = 0.0003). IS; in situ.</p

Significant difference of duration until recurrence between HPV-positive and -negative tumors.

<p>Fifty BC samples of the patients from whom information were available were compared between HPV-positive and -negative groups (a), and ER/HPV-double positive, ER-single positive, HPV single positive and ER/HPV-double negative groups (b). Numbers in brackets indicate total number of samples for each case, respectively. Student’s t-test was performed, with <i>p</i>-values indicated on the graph.</p

Abrogation of HPV-induced cancer phenotypes by shRNA against HPV E6, E7 and A3B.

<p>(a) A3B mRNA level in stable A3B, E6 or E7-knockdown MCF10A-HPV18 cells. A3B, E6 or E7 stably knocked down cells were established using shRNA retroviral vector. Total RNA was extracted from cells and then subjected to qRT-PCR. (b) γH2AX level in stable A3B-knockdown MCF10A-HPV18 cells. Cells were lysed after selection using puromycin and then subjected to western blot. The γH2AX and β-actin were detected using anti-γH2AX and β-actin Ab. The number at bottom of panel shows band intensity ratio after normalized by β-actin level. (c and d) HPV-18 E6 or E7 mRNA level in stable E6 or E7-knockdown MCF10A-HPV18 cells. Total RNA was extracted and then subjected to qRT-PCR. (e) γH2AX level in stable HPV18 E6 or E7-knockdown MCF10A- HPV18 cells. Cells were lysed after selection using puromycin, and then subjected to western blot. The γH2AX and β-actin were detected using anti-γH2AX and β-actin Ab. The number at bottom of panel shows band intensity ratio after normalization by β-actin level. Values represent the mean ± SD of at least three independent experiments. Student’s t-test was performed, with <i>p</i>-values indicated on the graph.</p