BRCA1 185delAG truncation protein, BRAt, amplifies caspase-mediated apoptosis in ovarian cells

Abstract

Breast and ovarian cancer patients with germline mutations in BRCA1 respond more favorably to initial chemotherapy. We previously reported that cells from women carrying the BRCA1 185delAG founder mutation undergo an enhanced caspase-3-mediated apoptotic response. Here, we report on the transient and stable transfection of cDNA coding for the putative truncated protein product of the BRCA1 185delAG mutant gene into BRCA1 wild-type human ovarian surface epithelial cells and ovarian cancer cells, resulting in cells with a heterozygous background containing two BRCA1 wild-type alleles and the BRCA1 185delAG transcript. The BRCA1 185delAG truncation (BRAt) protein did not alter epithelial cell morphology or induce tumorigenesis. However, upon treatment with staurosporine, BRAt cells showed increased levels of active caspase-3 and increased cleavage of caspase-3 substrates, PARP and DFF45. Additionally, XIAP and cIAP-1 protein are at reduced levels in untreated BRAt cells as compared to control cells. BRAt also reduced levels of phosphorylated Akt and overexpression of activated Akt in BRAt cells restored caspase-3 activity to that seen in wild-type cells. Further, BRAt expression increased chemosensitivity in platinum-resistant ovarian cancer cells. Taken together, our data demonstrate that truncated proteins arising from BRCA1 185delAG mutation increase Akt-mediated apoptosis, suggesting a possible mechanism by which ovarian cancer patients with this germline BRCA1 mutation may respond better to initial chemotherapy.

BRCA1 16 years later: risk‐associated BRCA1 mutations and their functional implications

“…To rule out the possible contribution of wild‐type BRCA1 haploinsufficiency to altered apoptosis in 185delAG cells, BRAT was expressed in wild‐type BRCA1 ovarian surface epithelial cells. In agreement with our earlier studies, BRAT enhanced caspase 3‐mediated apoptosis and diminished levels of phospho‐Akt, cIAP1 and XIAP [46]. In more recent studies, we found that BRAT upregulated the expression of maspin [47], a tumor suppressor important in apoptosis, invasion and metastasis that is uniquely overexpressed in several tumor types, including ovarian cancer [48].…”

BRCA1 185delAG Mutation Enhances Interleukin-1βExpression in Ovarian Surface Epithelial Cells

“…Previously, we demonstrated that human OSE cells with the BRAT mutation exhibited enhanced apoptosis and caspase-3 activation [ 11 ] as well as diminished levels of phosphorylated Akt, cellular inhibitor of apoptosis 1 (cIAP1), and X-linked inhibitor of apoptosis protein (XIAP) [ 12 ]. We also found that BRAT-mediated maspin expression was correlated with enhanced chemosensitivity [ 13 ] which is in agreement with clinical reports of increased survival in patients with elevated maspin levels [ 14 ]. Microscopic examination of ovarian specimens obtained following prophylactic oophorectomy from women with FH of OC indicated that greater than 85% presented with two or more abnormal OSE histologic features such as surface epithelial pseudostratification, surface papillomatosis, cortical invaginations of OSE, epithelial inclusion cysts, and epithelial hyperplasia [ 15 ].…”

“…The following SV 40-Large T-Antigen transfected human OSE (IOSE) cell lines were used: IOSE118 derived from a normal patient with a FH of breast and/or OC with wtBRCA1 status confirmed [ 13 ]; IOSE 121 derived from a normal patient with NFH of breast and/or OC though BRCA1 mutation status was not determined; and IOSE 3261-77 and IOSE 1816-686 derived from normal patients with a FH of breast and/or OC as well as confirmed carriers of the BRCA1 185delAG mutation. IOSE cells were cultured in Medium 199/MCDB 105 (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS) and gentamicin.…”

“…IOSE cells were cultured in Medium 199/MCDB 105 (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS) and gentamicin. Stable PCDNA (pcDNA3.1) and BRAT clones (BRATc1, BRATc2) were generated by transfection of 2.5 × 10 5 IOSE 118 cells with 2.5 μ g of pcDNA3.1 or Flag-BRAT with G418 resistance gene as previously described [ 13 ] using Program X-005, Kit V, and the Nucleofector device (Amaxa/Lonza, Walkersville, MD). Stable cells were maintained in 1 mg/mL G418 selection media and BRAT clones were confirmed to express BRAT by RT-PCR [ 13 ].…”

“…For BRAT semiquantitative PCR to confirm transfection, 1 μ g total RNA, oligo(dT), and reverse transcriptase were used to generate single-strand cDNA as previously described [ 13 ]. The cDNA samples were amplified using the Applied Biosystems GeneAmp RNA PCR Core Kit (Foster City, CA).…”