Oestrogen-mediated phosphorylation and stabilization of BRCA2 protein in breast.

Disease-associated BRCA2 mutations typically result in protein truncations that delete the phosphorylation-regulated S3291 BRCA2 domain that interacts with Rad51. BRCA2 hereditary breast cancers are usually ER(+), differing from BRCA1 hereditary cancers, which are usually ER(-). We studied BRCA2 protein expression and S3291 phosphorylation in normal breast tissues and in sporadic breast cancers and observed that BRCA2 is expressed and phosphorylated in normal breast and 10 ER(+) breast cancers but not in 10 ER(-) breast cancers. In order to study this correlation between ER and BRCA2 expression, we studied ER(+) breast cancer cell lines. We found that a rapid increase in BRCA2 S3291 phosphorylation occurs following 17-beta-oestradiol (E2) treatment. This increase seen in BRCA2 total and phospho-S3291 protein levels was found to be unaffected with cycloheximide pre-treatment, but decreased following tamoxifen, ICI 182,780 or roscovitine treatment. This suggests a requirement for ER and cdk (cyclin-dependent kinase) in mediating the increased protein levels. MCF7 cell cycle distribution analysis following E2, in both the presence and absence of roscovitine (a cdk inhibitor), did not demonstrate any changes during an 8 h period, which further supports our hypothesis that mitogenic effects of E2 are not predominant at early time points. Studies with MG132 proteasome inhibitor and siRNA to skp2 support a model in which skp2-mediated proteasomal degradation of BRCA2 rapidly degrades BRCA2 protein in the absence of hormone treatment, which likely inhibits this pathway. E2 was shown to improve survival of MCF7 cells upon radiation treatment and roscovitine partially reversed this effect. We have demonstrated that BRCA2 protein is specifically expressed in ER(+) breast cancers and are investigating a pathway that may show a link between E2 action and BRCA2 protein function in breast cancer.

Preclinical studies of a new generation retroviral vector for ovarian cancer BRCA1 gene therapy.

OBJECTIVE: The aim of this study was to determine the preclinical stability, toxicity, and efficacy of a second-generation complement-resistant retroviral BRCA1 vector, MFG-BRCA1, for ovarian cancer gene therapy. METHODS: MFG-BRCA1 was packaged in human 293 renal cells and manufactured and tested under cGMP conditions and is allowed for use in humans by the Food and Drug Administration. Vector stability studies were performed in mice and human serum by PCR analysis. Toxicity in the animals was assessed at necropsy, evaluating for histological signs of inflammation and organ damage. Tissue culture efficacy studies were performed on ovarian and breast cancer cells. Animal efficacy studies were conducted in female nu/nu mice. Mice were injected intraperitoneally with SKOV-3 ovarian cancer cells and tumors were allowed to grow for 4 weeks. Mice were treated intraperitoneally with MFG-BRCA1 or control vectors. Survival of animals was compared in the MFG-BRCA1 versus the control groups. RESULTS: MFG-BRCA1 was more stable in human serum than LXSN-BRCA1sv. Toxicity as demonstrated by an inflammatory peritonitis was minimal. Significantly fewer clones were obtained using the MFG-BRCA1 versus the MFG vector alone in both cell lines. Efficacy studies in animals of MFG-BRCA1 demonstrated a near threefold increase in survival over control vector and twofold increase compared to the first generation LXSN-BRCA1sv vector. CONCLUSION: The reengineered complement-resistant MFG-BRCA1 retroviral vector is more effective and more stable than the previous generation LXSN-BRCA1sv vector.

Gene therapy for carcinoma of the breast: Therapeutic genetic correction strategies.

Gene therapy is a therapeutic approach that is designed to correct specific molecular defects that contribute to the cause or progression of cancer. Genes that are mutated or deleted in cancers include the cancer susceptibility genes p53 and BRCA1. Because mutational inactivation of gene function is specific to tumor cells in these settings, cancer gene correction strategies may provide an opportunity for selective targeting without significant toxicity for normal nontumor cells. Both p53 and BRCA1 appear to inhibit cancer cells that lack mutations in these genes, suggesting that the so-called gene correction strategies may have broader potential than initially believed. Increasing knowledge of cancer genetics has identified these and other genes as potential targets for gene replacement therapy. Initial patient trials of p53 and BRCA1 gene therapy have provided some indications of potential efficacy, but have also identified areas of basic and clinical research that are needed before these approaches may be widely used in patient care.

Ovarian Cancer Gene Therapy with BRCA1-An Overview.

The human breast and ovarian cancer susceptibility gene BRCA1 is a tumorsuppressor gene which is mutated and lost in hereditary breast and ovarian cancer, and has both alleles mutated in approximately 10-15% of cases of sporadic ovarian cancer. Studies of chromosome loss in ovarian cancer show that at least one allele of the BRCA1 gene is lost or mutated in up to 70% of sporadic ovarian cancers. Although no sporadic breast cancers contain BRCA1 mutations, our published study shows that expression of the mRNA is decreased suggesting that the BRCA1 gene is altered quantitatively in sporadic cancer and qualitatively in hereditary cancer. Decreased expression of the BRCA1 gene may also be important in cases of sporadic ovarian cancer that do not have BRCA1 mutations. The majority of mutant alleles are nonsense and should produce truncated proteins, which are predicted to vary in length from 5% to 99% of full-length protein. A relatively small number of missense mutations have also been identified, most commonly in the predicted ring finger domain in the N-terminus. Patients with mutations in BRCA1 differ clinically from nonfamilial breast cancer because the breast cancer is earlier in onset and more highly associated with ovarian cancer. We have recently obtained data indicating that families with BRCA1 mutations in the 3' portion of the gene develop early-onset breast cancer, but do not develop ovarian cancer, suggesting that expression of a truncated BRCA1 protein may suppress ovarian tumors, but not breast tumors (1).

Ovarian cancer BRCA1 gene therapy: Phase I and II trial differences in immune response and vector stability.

Gene therapy with viral vectors has shown some promise in nude mice models and in initial Phase I trials of patients with extensive metastatic cancer. A Phase I clinical trial (D. L. Tait et al., Clin. Cancer Res., 3: 1959-1968, 1997) of ovarian cancer patients treated with i.p. retroviral LXSN-BRCA1sv gene therapy reported stable vector, minimal antibody response, and tumor reduction. We initiated a Phase II trial on patients with less extensive disease to evaluate vector pharmacokinetics, immune response, toxicity, and efficacy. Patients received a surgically implanted peritoneal catheter to administer infusions of vector, as well as to retrieve daily samples of peritoneal fluid for analysis. Ovarian cancer patients received four daily i.p. injections of LXSN-BRCA1sv vector therapy for three cycles, 4 weeks apart. Patient peritoneal fluid and plasma were analyzed extensively by PCR, Western blot, complement level (CH50), and chemical and hematological tests. Phase II patients showed no response, no disease stabilization, and little or no vector stability. Because of vector instability and rapid antibody development, which differed dramatically from the Phase I trial data, the trial was terminated after treatment of six patients. Immune system status appears to have played a major role in whether gene therapy was effective. Comparison of Phase I and II patients showed significant differences in tumor burden, immune system status, and response to BRCA1 gene therapy.

BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair.

The breast cancer predisposition genes, BRCA1 and BRCA2, are responsible for the vast majority of hereditary breast cancer. Although BRCA2 functions to help the cell repair double-stranded DNA breaks, the function of BRCA1 remains enigmatic. Here, we develop a human genetic system to study the role of BRCA1 in oxidative DNA damage. We show that human cancer cells containing mutated BRCA1 are hypersensitive to ionizing radiation. This hypersensitivity can be reversed by the expression of forms of BRCA1 that are not growth suppressing. Reversal of hypersensitivity requires the ring finger of BRCA1, its transactivation domain, and its BRCT domain. Lastly, we show that unlike BRCA2, BRCA1 does not function in the repair of double-stranded DNA breaks. Instead, it functions in transcription-coupled DNA repair (TCR). TCR ability correlated with radioresistance as cells containing BRCA1 showed both increased TCR and radioresistance, whereas cells without BRCA1 showed decreased TCR and radiosensitivity. These findings give physiologic significance to the interaction of BRCA1 with the basal transcription machinery.

Mitogen-activated protein kinase kinase 2 activation is essential for progression through the G2/M checkpoint arrest in cells exposed to ionizing radiation.

An increasing body of evidence suggests that mitogen-induced activation of the RAF/ERK signaling pathway is functionally separate from the stress-induced activation of the SEK/JNK/p38 signaling pathway. In general, stress stimuli strongly activate the p38s and the JNKs while only weakly activating ERK1 and ERK2. However, a number of independent groups have now shown that the RAF/ERK signaling pathway is strongly activated by ionizing radiation. In this work, we examine this paradox. We show that both mitogen-activated protein (MAP) kinase kinase 1 (MEK1) and MAP kinase kinase 2 (MEK2) are activated by ionizing radiation. Blockage of this activation through the use of dominant negative MEK2 increases sensitivity of the cell to ionizing radiation and decreases the ability of a cell to recover from the G2/M cell cycle checkpoint arrest. Blocking MEK2 activation does not affect double-strand DNA break repair, however. Although MEK1 is activated to a lesser extent by ionizing radiation, expression of a dominant negative MEK1 does not affect radiation sensitivity of the cell, the G2/M checkpoint of the cell, or double-strand break repair. Because ionizing radiation leads to a different cell cycle arrest (G2/M arrest) than that typically seen with other stress stimuli, and because we have shown that MEK2 can affect G2/M checkpoint kinetics, these results provide an explanation for the observation that the MEKs can be strongly activated by ionizing radiation and only weakly activated by other stressful stimuli.

Ovarian cancer gene therapy.

Retroviral-mediated delivery of BRCA1 gene therapy (LXN-BRCA1sv, a normal splice variant form of BRCA1) was tested extensively in mouse models. It was found to be effective in reducing tumor burden and to be minimally toxic. Twelve phase I clinical trial patients with recurrent or persistent epithelial ovarian cancer were treated with one to three cycles of intraperitoneal vector. There was minimal toxicity, four patients developed fevers (< 102.5 degrees F) and three had sterile peritonitis, which resolved within 48 hours. The vector was found to be fairly stable in some patients at 24 hours as well as transferred into and expressed in patient tissues. Stable disease was noticed in 8 of the 12 patients, suggesting that the peritoneal cavity may be an appropriate site for gene therapy.

Double-strand break repair deficiency and radiation sensitivity in BRCA2 mutant cancer cells.

BACKGROUND: The protein product of the BRCA2 gene mediates repair of double-strand breaks in DNA. Because a number of cancer therapies exert cytotoxic effects via the initiation of double-strand breaks, cancers comprised of cells carrying BRCA2 gene mutations may be more amenable to treatment with agents that cause such breaks. METHODS: We identified a human pancreatic adenocarcinoma cell line lacking one copy of the BRCA2 gene and containing a mutation (6174delT) in the remaining copy. In vitro and in vivo experiments were conducted with this cell line and with other carcinoma cell lines matched for similar genetic mutations, similar differentiation status, and/or similar carcinoma type to examine double-strand break repair, sensitivity to drugs that induce double-strand breaks, and radiation sensitivity. RESULTS: BRCA2-defective cells were unable to repair the double-strand DNA breaks induced by ionizing radiation. These cells were also markedly sensitive to mitoxantrone, amsacrine, and etoposide (drugs that induce double-strand breaks) (two-sided P = .002) and to ionizing radiation (two-sided P = .001). Introduction of antisense BRCA2 deoxyribonucleotides into cells possessing normal BRCA2 function led to increased sensitivity to mitoxantrone (two-sided P = .008). Tumors formed by injection of BRCA2-defective cells into nude mice were highly sensitive (>90% tumor size reduction, two-sided P = .002) to both ionizing radiation and mitoxantrone when compared with tumors exhibiting normal BRCA2 function. Histologic analysis of irradiated BRCA2-defective tumors showed a large degree of necrosis compared with that observed for control tumors possessing normal BRCA2 function. CONCLUSION: BRCA2-defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA.

Antisense c-myc retroviral vector suppresses established human prostate cancer.

Prostate cancer eventually becomes androgen resistant, resumes growth, and kills the patient. Characterization of genetic events that lead to androgen refractory prostatic neoplasia has revealed the frequent overexpression of c-myc and uncontrolled prostate cancer proliferation. A novel strategy to combat advanced prostate cancer utilized a replication incompetent retrovirus that contained the mouse mammary tumor virus (MMTV) promoter within the retroviral vector to allow transcription of antisense c-myc gene within target prostate tumor cells. The transduction of cultured DU145 cells by XM6:MMTV-antisense c-myc RNA retrovirus did not affect cell proliferation in culture, yet a single direct injection of MMTV-antisense c-myc viral media into established DU145 tumors in nude mice produced a 94.5% reduction in tumor size compared to tumors treated with control virus MTMV sense fos and untreated tumor by 70 days. Two animals in the antisense c-myc-treated group had complete regression of their tumors. Histopathological examination of the tumors revealed that MMTV-antisense c-myc-transduced DU145 tumors had increased tumor cell differentiation, decreased invasion, and a marked stromal response. The mechanism for the antitumor effect of MMTV-antisense c-myc retrovirus appears to be suppression of c-myc mRNA and protein, and decreased bcl-2 protein. The in vivo transduction of prostate cancer cells with MMTV-antisense c-myc retroviruses reduced tumor growth by suppressing c-myc, resulting in the down-regulation of bcl-2 protein. Consequently, the MMTV-antisense c-myc retrovirus may be useful for gene therapy against advanced, hormone-refractory prostate cancer.

Gene therapy for breast and ovarian cancer with BRCA1.

As an initial step toward gene therapy for ovarian cancer, we conducted a Phase 1 trial to assess the pharmacokinetics and toxicity of intraperitoneal BRCA1sv retroviral vector therapy. Gene transfer and expression were documented by PCR, southern blot, RT-PCR and nuclease protection assays. Pharmacokinetics were assessed by PCR and southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of twelve patients developed an acute sterile peritonitis which spontaneously resolved within 48 hours. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to four months. Eight patients showed stable disease for 4 to 16 weeks. Three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). Ovarian cancer may provide an imporant model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by intraperitoneal therapy.

Finkel-Biskis-Reilly mouse osteosarcoma virus v-fos inhibits the cellular response to ionizing radiation in a myristoylation-dependent manner.

DNA damage is recognized as a central component of carcinogenesis. DNA-damaging agents activate a number of signal transduction pathways that lead to repair of the DNA, apoptosis, or cell cycle arrest. It is reasoned that a cell deficient in DNA repair is more likely to acquire other cancer-promoting mutations. Despite the recent interest in the link between DNA damage and carcinogenesis, retroviral oncogenes have not yet been shown to affect the DNA damage-signaling pathway. In this report, we show that Finkel-Biskis-Reilly mouse osteosarcoma virus (FBR) v-fos, the retroviral homologue of the c-fos proto-oncogene, inhibits the cellular response to ionizing radiation. Cells that express FBR v-Fos show a decreased ability to repair DNA damage caused by ionizing radiation, and these cells show decreased survival in response to ionizing radiation. In addition, FBR v-Fos inhibits DNA-dependent protein kinase, a kinase specifically activated upon exposure to ionizing radiation. These effects were specific to ionizing radiation, as no effect of FBR v-Fos on the UV light signaling pathway was seen. Last, these effects were dependent on a lipid modification required for FBR v-Fos tumorigenesis, that of myristoylation of FBR v-Fos. A non-myristoylated mutant FBR v-Fos caused none of these effects. This study suggests that a retroviral oncogene can lead to an increased genomic instability, which can ultimately increase the carcinogenic potential of a cell.

Finkel-Biskis-Reilly osteosarcoma virus v-Fos inhibits adipogenesis and both the activity and expression of CCAAT/enhancer binding protein alpha, a key regulator of adipocyte differentiation.

Finkel-Biskis-Reilly (FBR) osteosarcoma virus v-Fos causes tumors of mesenchymal origin, including osteosarcomas, rhabdomyosarcomas, chondrosarcomas, and liposarcomas. Because the cell of origin in all these tumors is a pluripotent mesenchymal cell, the variety of tumors seen in mice which express FBR v-Fos implies that FBR v-Fos inhibits multiple differentiation pathways. To study the mechanism of FBR v-Fos' inhibition of mesenchymal differentiation, we utilized an in vitro model of adipocyte differentiation. We show by both morphological and biochemical means that FBR v-Fos inhibits adipocyte differentiation in vitro. This inhibition is due to FBR v-Fos' inhibition of the growth arrest characteristic of terminal differentiation and FBR v-Fos' inhibition of the expression and activity of a key regulator of this growth arrest, C/EBPalpha. The in vitro inhibition of adipogenesis by FBR v-Fos has in vivo significance as immunostaining of FBR v-Fos-induced tumors shows no CCAAT/enhancer binding protein (EBP)-alpha expression. These data implicate C/EBPalpha as a protein involved in the generation of liposarcomas.

A phase I trial of retroviral BRCA1sv gene therapy in ovarian cancer.

Gene transfer of BRCA1sv (a normal splice variant of BRCA1) into ovarian cancer cells produces growth inhibition in vitro and tumor suppression in nude mouse xenografts. As an initial step toward gene replacement therapy for ovarian cancer, we conducted a Phase I trial to assess the pharmacokinetics and toxicity of i.p. BRCA1sv retroviral vector therapy. Following placement of an indwelling Port-a-Cath in patients, a dose escalation study was performed of four daily i.p. infusions spanning doses from 3 to 300 ml (i.e., 10(10) viral particles) at half-log intervals (23 cycles in 12 patients). Gene transfer and expression were documented by PCR, Southern blot, reverse transcription-PCR, and nuclease protection assays. Pharmacokinetics were assessed by PCR and Southern blots detecting vector DNA, and toxicity was evaluated by clinical exam and fluid analysis. Three of 12 patients developed an acute sterile peritonitis, which spontaneously resolved within 48 h. Plasma and peritoneal antibodies to the retroviral envelope protein were detected only in patients treated with the highest dose levels but not in others, despite repeat dosing for an interval of up to 4 months. Eight patients showed stable disease for 4-16 weeks, and three patients showed tumor reduction with diminished miliary tumor implants at reoperation (two patients) and radiographic shrinkage of measurable disease (one patient). The vector-related complication of peritonitis was observed in three patients but resolved quickly as in preclinical mouse studies. Ovarian cancer may provide an important model for retroviral gene therapy studies due to vector stability, minimal antibody response, and access to tumor by i.p. therapy.

Breast cancer genes: therapeutic strategies.

Although effective treatments for breast cancer predated the identification of causative molecular defects in humans, it is widely hoped that an understanding and/or manipulation of the key genetic events will lead to even more effective therapies or even cures. Powerful methods of positional cloning and gene identification have identified the breast cancer genes, BRCA1 and BRCA2, which together are responsible for the majority of cases of hereditary breast and ovarian cancer. Although the BRCA1 gene is rarely mutated in sporadic breast or ovarian cancer, levels of BRCA1 mRNA and protein are markedly decreased in the majority of sporadic cases of cancer. This suggests that hereditary and sporadic breast cancer share common genetic themes and that treatments aimed at increasing levels of BRCA1 or BRCA2 may be useful for both hereditary and sporadic cancers. We have demonstrated that gene transfer of wild-type BRCA1 inhibits the growth of sporadic breast and ovarian cancer cells and suppresses growth of established breast and ovarian tumor models in nude mice. Mutant BRCA1 genes do not inhibit growth or suppress tumor, providing additional evidence that BRCA1 is a tumor-suppressor gene. Strategies designed to increase BRCA1 expression or development of BRCA1-mimetic agents may be ultimately useful as therapeutic approaches.

Myristylation of FBR v-fos dictates the differentiation pathways in malignant osteosarcoma.

Myristylation of FBR v-fos, a c-fos retroviral homologue that causes osteosarcomas in mice, determines many of its transcriptional properties in vitro. To determine whether myristylation of FBR v-fos contributes to in vivo tumorigenicity, we examined its transforming capability in comparison to a nonmyristylated FBR v-fos (G2A-R). Retroviral infections with FBR v-fos and G2A-R transform BALB/c-3T3 cells. The number, size, and cellular morphology of foci generated by both FBR and G2A-R are indistinguishable. However, marked biological differences were found in transgenic mice expressing either the myristylated FBR v-fos or the nonmyristylated G2A-R. 11 of 26 FBR v-fos transgenic mice died as a result of gross tumor burden. None of the 28 G2A-R transgenic mice died from tumor burden, and only two of the G2A-R mice developed bone tumors. Histologic examination of the tumors reveals that the FBR v-fos bone tumors contain malignant cells with features of four cell lineages (osteocytes, chondrocytes, myocytes, and adipocytes) in an environment rich in extracellular matrix (ECM). However, the G2A-R tumors exist in an environment devoid of ECM and display malignant cells with features of adipocytes. Masson staining reveals that the ECM of the FBR tumors stains strongly for collagen. Immunohistochemical staining with collagen III antibody demonstrates an abundance of collagen III expression in this ECM. While NH2-terminal myristylation is not required for FBR immortalization and transformation, it is essential in determining the degree of differentiation and tumorigenicity of malignant cells.

Growth retardation and tumour inhibition by BRCA1.

Inherited mutations in BRCA1 predispose to breast and ovarian cancer, but the role of BRCA1 in sporadic breast and ovarian cancer has previously been elusive. Here, we show that retroviral transfer of the wild-type BRCA1 gene inhibits growth in vitro of all breast and ovarian cancer cell lines tested, but not colon or lung cancer cells or fibroblasts. Mutant BRCA1 has no effect on growth of breast cancer cells; ovarian cancer cell growth is not affected by BRCA1 mutations in the 5' portion of the gene, but is inhibited by 3' BRCA1 mutations. Development of MCF-7 tumours in nude mice is inhibited when MCF-7 cells are transfected with wild-type, but not mutant, BRCA1. Most importantly, among mice with established MCF-7 tumours, peritoneal treatment with a retroviral vector expressing wild-type BRCA1 significantly inhibits tumour growth and increased survival.