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This approach is best suited to students who already are familiar with the basic content but need experience applying it to clinical cases or problems. After studying assigned podcasts or readings the students attend an interdisciplinary session focused on a specific clinical presentation.

The Commonwealth Medical College (TCMC) accepted its first class of students in 2009. Being a new medical school gave a group of pioneering faculty members a unique opportunity, to develop a curriculum unencumbered by tradition. The Clinical Presentation Curriculum model provided the framework for an integrated course which utilized podcasts and active learning techniques to facilitate student learning using a flipped classroom approach. In this monograph, we describe how we developed the TCMC model of an integrated system course for the second year medical students reflecting on our 5 years’ experience with this integrated and flipped classroom model.

Learning objectives are a common learning guide but little is known regarding which students may benefit from their use. We have previously reported that male medical students benefit from the use of learning objectives more than female students. We next tested whether specific Myers-Briggs type indicator (MBTI) benefitted from using these objectives. Student’s MBTI types were determined by standard methods, and student use of online learning objectives was tracked in Blackboard. We then determined which types of students benefitted from learning objective use based on course exams and a standardized test. Although all MBTI types who downloaded objectives except intuitives (I) had better course exam scores, only sensing (S) types had enough benefit to significantly affect standardized test scores. Sensing types who downloaded learning objectives had a 30 % gain in score over non-downloaders on the course exam (80.7 ± 1.3 versus 68.7 ± 1.3; P = 0.001). The sensing–intuitive axis appears dominant over gender since sensing-type females benefit but intuitive-type males do not. MBTI type does not appear to affect the decision to download learning objectives as there is no significant difference in objective downloading rates between types. Students with specific Myers-Briggs types, particularly sensing, benefit from downloading learning objectives more than other students. These results support the notion that multiple learning methods may be needed to teach students with diverse learning needs.

Few studies have analyzed the relationship between exam performance and learning objectives use in medical education. We hypothesized that students who viewed learning objectives would benefit in exam performance. Our curriculum is based on students’ learning content through individually viewed podcasts followed by in-class activities allowing deliberate practice and applications. We utilized the tracking feature in Blackboard to identify which second-year medical students (MD2) downloaded topic-specific learning objectives prior to a learning objective-based block exam testing higher-order subject knowledge. We compared end-of-block exam scores and comprehensive basic science self-assessment (CBSSA) scores of students who downloaded learning objectives (downloaders) with those who did not (non-downloaders). Learning objective downloaders had higher mean block exam scores (79.7 % ± 1.09 SEM; n = 39) than non-downloaders (71.0 % ± 1.77 SEM; n = 24; p = 0.0001), but men had a greater benefit than women (mean difference for men 10.1 % (p = .0004) versus 7.0 % for women (p = .03)), even though they had similar downloading rates (men = 55 % and women = 70 %; p = 0.30). Students were encouraged to use learning objectives in a subsequent block and most did, with downloading rate increasing significantly from 39/64 (60.9 %) to 54/64 (84.4 %; p = 0.005). However, only men benefitted from this new use of learning objectives (mean 12.9 versus 4.7; p = 0.0044). CBSSA test analysis also showed that men who were downloaders had 30 % higher scores than those who were not (p = 0.02), but women showed no significant difference (p = 0.54). Downloading learning objectives increases academic performance on both course and standardized exams but predominantly benefits men.

Purpose Mutations in the BRCA2 gene are dominantly inherited but cause cancers when the wild-type allele has loss of heterozygosity (LOH) within the cancer. Because most disease-associated BRCA2 mutations are protein-truncating mutations, a test for truncated BRCA2 proteins should identify most BRCA2 hereditary cancers. Methods We have developed a tissue truncation test to identify truncated BRCA2 proteins in breast cancer tissue biopsies in vivo that does not use amplification or genetic manipulations. N-terminal and C-terminal antibodies are used to visualize protein truncation by demonstrating that the beginning of the protein is present but the end (ie, terminus) is absent. Results A quantitative C-terminal immunostaining score or a C-terminal to N-terminal truncation ratio correctly classified 20 of 21 breast cancers arising in BRCA2 mutation carriers and 57 of 58 cancers arising outside the context of a multiple-case breast cancer family. This represents a sensitivity of 95% and a specificity of 98%. Because of the presence of C-terminal BRCA2 protein and atypical clinical features of the misclassified cancer in a BRCA2 mutation carrier, we performed polymerase chain reaction and sequence analyses on this cancer. The results showed continued presence of the BRCA2 wild-type allele in the cancer, which indicated that intact BRCA2 protein was present in this cancer. Conclusion This immunohistochemistry-based test (which takes only 4 hours) appears to identify BRCA2 hereditary cancer with high accuracy. The test also appears to diagnose the biochemical loss of BRCA2 protein in cancers (ie, BRCA2-mutant genotype), which will usually but not always agree with the presence of a germline BRCA2 mutation found by susceptibility testing by DNA sequencing of blood samples.

Mutations in the BRCA2 gene are associated with inherited, early-onset breast cancer. CAPAN-1 cells have been very beneficial in studying how BRCA2 mutations contribute to malignant transformation. They exhibit loss of heterozygosity (LOH), and the remaining copy of BRCA2 has a 6174delT mutation, which causes a premature C-terminal truncation that removes the domains for DNA repair and the nuclear localization signals. The DNA repair protein RAD51, which interacts with BRCA2, exhibits impaired nuclear translocation in CAPAN-1. It has been speculated that RAD51 may require BRCA2 for nuclear entry and C-terminally truncated BRCA2 may retain RAD51 in the cytoplasm. This may cause heterozygous individuals to exhibit deficient DNA repair and cell viability comparable to individuals with LOH or bi-allelic BRCA2 mutations. We simulated a heterozygous condition by employing stably transfected CAPAN-1 cells with wildtype BRCA2. Our studies determined that fusion of a nuclear localization signal to RAD51 did not increase its ability to independently enter the nuclei of CAPAN-1 cells. Furthermore, restoration of functional BRCA2 did not significantly improve DNA repair, or re-establish cell viability in CAPAN-1 cells. The results imply that C-terminally truncated BRCA2 hinders RAD51 nuclear translocation, possibly contributing to genetic instabilities in homozygous, as well as heterozygous individuals.

Brown, E. T., Robinson-Benion, C. and Holt, J. T. Radiation Enhances Caspase 3 Cleavage of Rad51 in BRCA2-Defective Cells. Radiat. Res. 169, 595–601 (2008). After DNA damage, caspases cleave and activate proteins involved in cell death by apoptosis but also cleave and inactivate proteins implicated in DNA repair. Here we report a rapid onset of Rad51 cleavage by caspase 3 in BRCA2-defective mouse and human cells. This rapid cleavage was reduced markedly by transfer of full-length human BRCA2 into BRCA2-defective mouse or human cells, which also blocked the association of caspase 3 and Rad51 proteins. Overall caspase 3 activity was increased in BRCA2-defective cells, but the time course was much slower than that for Rad51 cleavage. We further showed that caspase 3 cleavage of Rad51 resulted in a functional decrease in Rad51 strand exchange activity and that inhibition of caspase 3 activity increased Rad51 protein levels and Rad51 foci. These findings indicate that BRCA2 inhibits Rad51 cleavage and...

To characterize the role of BRCA1 in mammary gland development and tumor suppression, a transgenic mouse model of BRCA1 overexpression was developed. Using the mouse mammary tumor virus (MMTV) promoter/enhancer, transgenic mice expressing human BRCA1 or select mutant controls were generated. Transgenic animals examined during adolescence were shown to express the human transgene in their mammary glands. The mammary glands of 13-week-old virgin homozygous MMTV-BRCA1 mice presented the morphology of moderately increased lobulo-alveolar development. The mammary ductal trees of both hemizygous and homozygous MMTV-BRCA1t340 were similar to those of control non-transgenic littermates. Interestingly, both hemi- and homozygous mice expressing a splice variant of BRCA1 lacking the N-terminal RING finger domain (MMTV-BRCA1sv) exhibited marked mammary lobulo-alveolar development, particularly terminal end bud proliferation. Morphometric analyses of mammary gland whole mount preparations were used to measure epithelial staining indices of ~35% for homozygous MMTV-BRCA1 mice and ~60% for both hemizygous and homozygous MMTV-BRCA1sv mice versus ~25% for non-transgenic mice. Homozygous MMTV-BRCA1 mice showed delayed development of tumors when challenged with 7,12 dimethylbenzanthracene (DMBA), relative to non-transgenic and homozygous BRCA1t340 expressing mice. In contrast, homozygous MMTV-BRCA1sv transgenic animals were sensitized to DMBA treatment and exhibited a very rapid onset of mammary tumor development and accelerated mortality. MMTV-BRCA1 effects on mortality were restricted to DMBA-induced tumors of the mammary gland. These results demonstrate in vivo roles for BRCA1 in both mammary gland development and in tumor suppression against mutagen-induced mammary gland neoplasia.

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.

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 BRCA2defective 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: BRCA2defective cancer cells are highly sensitive to agents that cause double-strand breaks in DNA. [J Natl Cancer Inst 1998;90: 978‐85]

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.

By reporting on the possible existence of a hereditary cancer and recommending further genetic evaluation, the pathologist can help identify families with hereditary cancer. Currently, however, recognition of a case of hereditary cancer relies predominantly on clinical criteria, as common cancers show only a few specific pathologic features that suggest hereditary predisposition. Furthermore, clinical criteria alone often fail to identify mutant gene carriers. In contrast to genes that have a high penetrance for causing cancer, such as BRCA1, APC, RET, and the mismatch repair genes, identifying carriers of mutant genes having a lower penetrance (as is the case for the BRCA2 gene) is more difficult. Studies to identify markers for mutant germline cancer-predisposing genes will typically require studying archival tissue specimens, but germline genetic testing on archival samples for research purposes is complicated by the need for patient consent unless specimens are stripped of their patient identifiers. Pathologists contemplating such research should seek informed consent for genetic testing prior to archiving tissues. The need for consent has led to concerns that certain types of cancer research projects will no longer be feasible; if patient consent has not been obtained, however, institutional review board-approved protocols that facilitate obtaining patient clinical data while maintaining patient confidentiality can overcome this problem. As clinical genetic testing for hereditary cancer becomes part of clinical practice, there are many concerns about the quality of service at each step in the process. One of the most important requirements for quality is to ensure that the performance of a genetic test is always linked to adequate pretest and posttest genetic counseling. Reporting of genetic test results should be standardized and include information about the technique used to identify mutations, the sensitivity and specificity of the test in detecting such mutations, and the limitations of the test. The report should also contain recommendations for further action and information packets to assist in the interpretation of the test. Pathologists have expertise in the area of laboratory proficiency testing and can play an important role in the establishment of a national proficiency program to ensure a quality genetic testing process. Cancer 1997; 80:636-48. © 1997 American Cancer Society.

Scientific Abstract Although murine retroviral vectors can transduce diverse types of proliferating cells, inclusion of tissue-specific or inducible promoters within retroviral vectors should theor...

Germline mutations in BRCA1 are responsible for most cases of inherited breast and ovarian cancer. However, the function of the BRCA1 protein has remained elusive. We now show that BRCA1 encodes a 190-kD protein with sequence homology and biochemical analogy to the granin protein family. Interestingly, BRCA2 also includes a motif similar to the granin consensus at the C terminus of the protein. Both BRCA1 and the granins localize to secretory vesicles, are secreted by a regulated pathway, are post-translationally glycosylated and are responsive to hormones. As a regulated secretory protein, BRCA1 appears to function by a mechanism not previously described for tumour suppressor gene products.

Histopathologic study of human breast biopsy samples has identified specific lesions which are associated with a high risk of development of invasive breast cancer. Presumably, these lesions (collectively termed premalignant breast disease) represent the earliest recognizable morphologic expression of fundamental molecular events that lead to the development of invasive breast cancer. To study molecular events underlying premalignant breast disease, we have developed a method for isolating RNA from histologically identified lesions from frozen human breast tissue. This method specifically obtains mRNA from breast epithelial cells and has identified three genes which are differentially expressed in premalignant breast epithelial lesions. One gene identified by this method is overexpressed in four of five noncomedo ductal carcinoma in situ lesions and appears to be the human homologue of the gene encoding the M2 subunit of ribonucleotide reductase, an enzyme involved in DNA synthesis.

Viral oncogenes are generally believed to cause transformation through disregulated mimicry of their cellular homologues. However, here we show that FBR v-fos, unlike c-fos, transcriptionally activates unique genes in retrovirally induced chondro-osseous sarcomas. We show that FBR v-fos transactivates the collagen III and stromelysin promoters and that the unique transcriptional properties of transforming FBR depend upon its N-terminal myristylation and the differentiation state of the cell. Deletion or mutation of the myristylation site results in a loss of FBR v-fos transactivation in HeLa and undifferentiated 3T3-L1 preadipocyte cell lines. FBR v-fos transactivation of collagen III maps to a negative regulatory site which binds a key regulator of adipocyte differentiation, C/EBP alpha. Cotransfection of C/EBP alpha abolishes FBR v-fos transactivation of the alpha 1(III) collagen promoter. Furthermore, FBR v-fos cannot transactivate collagen III subsequent to adipocyte differentiation. We also demonstrate that collagen III transcription is reduced during adipocyte differentiation as the transcriptional activity of C/EBP alpha is concomitantly induced. Our results indicate that FBR v-fos transactivation depends upon its cotranslational myristylation and maps to a negative regulatory region which binds C/EBP alpha.

Mutations within the tumor suppressor BRCA1 cause the majority of hereditary breast and ovarian cancers. The BRCA1 protein is an important regulator of DNA double-strand break repair, and BRCA1-deficient cells are highly sensitive to ionizing radiation. Furthermore, BRCA1 function may contribute to enforcement of the G(2) cell cycle checkpoint. E3-ubiquitin ligase activity is the only known enzymatic activity of BRCA1, which is mediated by the N-terminal RING finger domain. The C-terminal BRCT repeat domain, which mediates protein-protein interactions, is the only other identified structural domain. By investigating cancer-linked mutations within each domain, we demonstrate that truncation of the BRCT domain greatly impairs the stability and nuclear localization of BRCA1 protein. A missense mutation within the RING domain does not affect these biochemical properties. However, both mutant forms of BRCA1 fail to colocalize in nuclear foci with the known BRCA1-interacting proteins BARD1 and BACH1, which are important for DNA repair. This failure occurs despite the continued ability of the RING mutant protein to interact with BACH1 and the ability of the BRCT mutant to interact with BARD1. Furthermore, neither mutant form of BRCA1 is recruited into DNA damage-associated foci marked by gamma-H2AX. Therefore, our data suggest that both the RING and BRCT domains of BRCA1 are required for an early step in the function of BRCA1 during DNA repair: recruitment to the sites of DNA damage.

BRCA1, with its binding partner BARD1, regulates the cellular response to DNA damage in multiple tissues, yet inherited mutations within BRCA1 result specifically in breast and ovarian cancers. This observation, along with several other lines of evidence, suggests a functional relationship may exist between hormone signaling and BRCA1 function. Our data demonstrates that AKT activation promotes the expression of BRCA1 in response to estrogen and IGF-1 receptor signaling. Further, we have identified a novel AKT phosphorylation site in BRCA1 at S694 which is responsive to activation of these signaling pathways. This rapid increase in BRCA1 protein levels appears to occur independently of new protein synthesis and treatment with the clinically utilized proteasome inhibitor bortezomib similarly leads to a rapid increase in BRCA1 protein levels. Together, these data suggest that AKT phosphorylation of BRCA1 increases total protein expression by preventing proteasomal degradation. AKT activation also appears to support nuclear localization of BRCA1, and co-expression of activated AKT with BRCA1 decreases radiation sensitivity, suggesting this interaction has functional consequences for BRCA1's role in DNA repair. We conclude that AKT regulates BRCA1 protein stability and function through direct phosphorylation of BRCA1. Further, the responsiveness of the AKT-BRCA1 regulatory pathway to hormone signaling may, in part, underlie the tissue specificity of BRCA1 mutant cancers. Pharmacological targets within this pathway could provide strategies for modulation of BRCA1 protein, which may prove therapeutically beneficial for the treatment of breast and ovarian cancers.

A brief exposure of quiescent, density-arrested Balb/c 3T3 fibroblasts to platelet-derived growth factor (PDGF) results in expression of the proto-oncogene c-fos; furthermore, the translation product of c-fos, p55fos, was shown to have increased stability in cells upon continued exposure to PDGF. The induction of competence or growth initiation requires a longer exposure to PDGF than that necessary for the induction of the immediate-early, growth-related genes. The need for the continued presence of PDGF for growth initiation beyond the time required for the induction of immediate-early gene expression may be due, in part, to PDGF-dependent post-translational stabilizations of gene products. We speculate that a PDGF-mediated event increases p55fos stability, resulting in a continued elevated level of Fos protein, which in turn allows a continued Fos-mediated activity required by Balb/c 3T3 cells to become competent to enter the cell cycle.

We have employed antisense methods to study the transcriptional functions of c-fos protein (Fos). Clones expressing inducible anti-fos RNA have been employed to inhibit c-fos expression, resulting in activation of c-fos transcription by inhibiting its normal repressor function. The sites of negative regulation by Fos have been mapped using this antisense mapping method which demonstrates that the serum response element represents the major site of repression by endogenous c-fos protein. A similar strategy (antisense cloning) has been employed to clone four target genes that are Fos dependent. These cDNAs encode mRNAs that are rapidly induced by serum (although this induction is blocked by cycloheximide) but are blocked by induction of anti-fos RNA. These inhibitory methods of studying transcription factor function are extremely useful for transcription factors (like Fos) that require cooperation with other factors to modulate gene transcription.

Breast cancers with BRCA2 mutations exhibit DNA repair defects and are particularly sensitive to radiation. BRCA2 interacts with Rad51 in a complex manner involving internal BRC and C-terminal TR2 domains which play a key role in homologous recombination. BRCA2 expression also modulates Rad51 protein levels such that Rad51 protein is relatively decreased in BRCA2-defective cancer cells. This is mediated in part through BRCA2's capacity to protect Rad51 from caspase-3 proteolytic degradation. In order to distinguish between functional and expression related roles for BRCA2 we studied the results of Rad51 overexpression in mouse and human cells with inactivating BRCA2 mutations. The results show that overexpression of wild-type Rad51 partially rescues BRCA2 deficiency but that overexpression of a caspase-3 resistant Rad51 completely complements the BRCA2 defect in radiation responsiveness. These results indicate that Rad51 can compensate for some aspects of a BRCA2 gene defect and suggest that Rad51 expression levels may be an important modifier of the BRCA2 defective genotype.

The Hereditary Cancer Program at Vanderbilt-Ingram Cancer Center (VICC) was established in mid-2012 and provides cancer genetic services to patients and family members who are at risk for family cancer syndromes. During this time, the testing paradigm has markedly shifted from testing a small number of genes to a larger multi-gene set. HYPOTHESIS AND METHODS: We hypothesized that multi-gene testing would identify a higher rate of pathogenic mutations in breast cancer patients than the standard BRCA1/2 testing paradigm. To test this notion, we examined the records of 641 women with breast cancer seen in our clinic from July 2012 through Dec 2014 and tabulated the test outcome in women tested for BRCA1/2 only and women who had multi-gene panels. Patient characteristics were compared between the two groups. RESULTS: Excluding 17 (3%) women with a known familial mutation and the 127 (20%) women who did not proceed with testing, 497 women had usual Sanger BRCA1/2 (189; 38%) or a multi-gene NGS testing (308; 62%). 40 (13%) women were found to have a pathogenic mutation using the multi-gene panel compared to 13 (6%) women who had a restricted BRCA1/2 sequencing (P=0.035, Fishers Exact Test). The 13 women with Sanger BRCA1/2 were younger at diagnosis (40.6 vs 48 yrs) and more likely to have triple negative (TN) disease (38% vs 18%) compared to the 40 women diagnosed with multi-gene panels. TN disease was not confined to BRCA1 carriers, however, as 3 of 7 TN patients had a mutation in BRCA2, PALB2, and ATM, respectively. Thus, 29% (2/7) of our triple negative patients would not have been identified without multi-gene panels. In addition to BRCA1 (6; 15%) and BRCA2 (5; 12.5%), 6 women had mutations in ATM (15%), 7 in CHEK2 (17.5%), 6 in MUTYH (15%), 4 in PALB2 (10%), 2 in TP53 (5%), and 1 each in FANCC, PMS2, RAD51D and XRCC2 (2.5% each). 105 patients (35%) who did not have a deleterious mutation on a multi-gene panel were found to have one or more variants of uncertain significance (VUS) compared to 4 patients who underwent BRCA1/2 testing alone (4/189; 2%). There was a significant difference between providers when ordering hereditary cancer testing, with MD or NP ordering panel testing at a greater rate compared to Genetic Counselors (72% vs 53%; P< 0.0001). CONCLUSION: We have examined the outcomes of genetic tests for 497 women with breast cancer during a time of great change in the approach to testing. Our study supports the paradigm that multi-gene panels will identify additional pathogenic mutations in genes other than BRCA1/2, which could increase clinical efficiency and improve patient outcomes. However, our study also found a high VUS rate in this group of patients, which will require additional clinical time to track for potential changes in pathogenicity. Future studies will focus on potential differences in management for patients found to have alterations on multi-gene tests. Citation Format: Wiesner GL, Lewis S, Holt J, Morgan RH, Riddle DA, Trump SA, McReynolds K. The changing paradigm of hereditary cancer testing: Comparison of tests in 497 women with breast cancer evaluated at an NCI designated cancer center. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P6-06-01.

Transforming growth factor beta 1 (TGF-beta 1) is a potent growth inhibitor for many cell types, including most epithelial cells. However, the mechanism of growth inhibition is unknown. In skin keratinocytes, TGF-beta 1 has been shown to inhibit growth and to rapidly reduce c-myc expression. It has been demonstrated that protein synthesis is required for TGF-beta 1 regulation of c-myc in keratinocytes. Here we present evidence that treatment of mouse BALB/MK keratinocyte cells with either antisense c-myc oligonucleotides or TGF-beta 1 inhibited cell entry into S phase. These results suggest that TGF-beta inhibition of c-myc expression may be essential for growth inhibition by TGF-beta 1. The block in c-myc expression by TGF-beta 1 occurred at the level of transcriptional initiation. Studies with a series of 5' deletion c-myc/chloramphenicol acetyltransferase constructs indicated that a cis regulatory element(s), which resides between positions -100 and +71 relative to P1 transcription start site, is responsible for the TGF-beta 1 responsiveness. Based on these data, it is proposed that the mechanism of TGF-beta 1 growth inhibition involves synthesis or modification of a protein that may interact with a specific element(s) in the 5' regulatory region of the c-myc gene, resulting in inhibition of transcriptional initiation.

Mutations in the breast cancer susceptibility gene 1 (BRCA1) account for a substantial percentage of familial breast and ovarian cancers. Although BRCA1 is thought to function within the nucleus, it has also been located in the cytoplasm. In addition, BRCA1 accumulates in the nucleus of cells treated with leptomycin B, an inhibitor of chromosome region maintenance 1-mediated nuclear export, indicative of its active nuclear export via this pathway. The nuclear export signal in BRCA1 has been described as consisting of amino acid residues 81-99. However, a number of other tumor suppressors have multiple nuclear export sequences, and we sought to determine whether BRCA1 did also. Here, we report that BRCA1 contains a second nuclear export sequence that comprises amino acid residues 22-30. By use of the human immunodeficiency virus-1 Rev complementation assay, this sequence was shown to confer export capability to an export-defective Rev fusion protein. The level of export activity was comparable with that of residues 81-99 comprising the previously reported nuclear export sequence in BRCA1. Mutation of leucine 28 to an alanine reduced nuclear export by approximately 75%. In MCF-7 cells stably transfected with a BRCA1 cDNA containing mutations in this novel sequence or the previously reported export sequence, BRCA1 accumulated in the nucleus. These data imply that BRCA1 contains at least two leucine-dependent nuclear export sequences.

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.

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).

Exposure of HepG2 cells to nonsteroidal anti-inflammatory drugs (i.e., indomethacin and ibuprofen; NSAIDs) as well as resveratrol, caused increased expression of the mRNAs coding for the catalytic (Gclc) and modifier (Gclm) subunits of the glutathione synthetic enzyme, gamma-glutamylcysteine synthetase. In addition, indomethacin exposure increased intracellular glutathione content as well as inhibited glutathione depletion and cytotoxicity caused by diethyl maleate. Indomethacin-induced increases in the expression of gamma-glutamylcysteine synthetase mRNA were preceded by increases in steady state levels of intracellular pro-oxidants and glutathione disulfide accumulation. Simultaneous incubation with the thiol antioxidant N-acetylcysteine (NAC) inhibited indomethacin-mediated increases in GCLC mRNA, suggesting that increases in GCLC message were triggered by changes in intracellular oxidation/reduction (redox) reactions. Indirect immunofluorescence using intact cells demonstrated that indomethacin induced the nuclear translocation of Nrf2, a transcription factor believed to regulate GCLC expression. Immunoprecipitation studies showed that indomethacin treatment also inhibited Nrf2 tethering to KIAA0132 (the human homolog of Keap1 accession #D50922), which is believed to be a negative regulator of Nrf2. Consistent with this idea, over-expression of Nrf2 increased GCLC reporter gene expression and over-expression of KIAA0132 inhibited GCLC reporter gene activity as well as inhibited indomethacin-induced increases in the expression of GCLC. Finally, simultaneous treatment with NAC inhibited both indomethacin-induced release of Nrf2 from KIAA0132 and indomethacin-induced nuclear translocation of Nrf2. These results demonstrate that NSAIDs and resveratrol cause increases in the expression of gamma-glutamylcysteine synthetase mRNA and identify these agents as being capable of stimulating glutathione metabolism. These results also support the hypothesis that indomethacin-induced transcriptional activation of GCLC involves the redox-dependent release of KIAA0132 from Nrf2 followed by the nuclear translocation of Nrf2.

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.

Publisher Summary Functional analysis of human cancer susceptibility genes has been greatly facilitated by the use of gene inhibition methods, including homologous recombination in mice and antisense methodologies in human cells. Although homologous recombination has clear-cut advantages over antisense inhibition methods in mouse experimental models, antisense oligonucleotide approaches continue to serve as a rapid and important means of gene inhibition in human cell lines and are of great importance for the study of poorly conserved genes with different functions between the mouse and the human. Breast cancer susceptibility genes are examples of genes with different functions in mouse versus human model systems. Mouse breast cancer 1 ( BRCA1 ) is only 57% homologous to human BRCA1 , and BRCA1 functions differently in the two systems. BRCA1 is a powerful growth suppressor in both yeast and human systems but is required for cellular proliferation during mouse development. Humans heterozygous for BRCA1 mutations develop breast cancer early in life, but mice heterozygous for BRCA1 mutations do not develop cancers. Well-described differences in deoxyribonucleic acid (DNA) repair between mouse and human cells complicate the ability to extrapolate results from mouse models to patients. Mouse and human cells show differences in the amount of damage sustained per given DNA-damaging dose in the kinetics of DNA repair and in cellular survival at a given dose of a DNA-damaging agent. BRCA2 is also poorly conserved between the mouse and the human, although some clear functional similarities have been described.

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.

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.

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.

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.

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.

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.

Antisense oligonucleotides can have dramatic effects, but a recent paper suggests that the mechanism isn't always based on nucleotide sequence interactions.

We have characterized expression of the familial breast and ovarian cancer gene, BRCA1, in cases of non-hereditary (sporadic) breast cancer and analyzed the effect of antisense inhibition of BRCA1 on the proliferative rate of mammary epithelial cells. BRCA1 mRNA levels are markedly decreased during the transition from carcinoma in situ to invasive cancer. Experimental inhibition of BRCA1 expression with antisense oligonucleotides produced accelerated growth of normal and malignant mammary cells, but had no effect on non-mammary epithelial cells. These studies suggest that BRCA1 may normally serve as a negative regulator of mammary epithelial cell growth whose function is compromised in breast cancer either by direct mutation or alterations in gene expression.

Publisher Summary This chapter focuses on the strategies and controls that can limit artifactual results: in particular, the use of reversal or rescue methods to hybrid arrest the antisense effect. The ability to manipulate gene expression within mammalian cells has provided powerful experimental approaches for studies of gene function and gene regulation. Methods that inhibit gene expression are particularly important because they permit studies probing the normal function of a specific gene product within a cell. Antibody injection and antisense studies have shown that cellular protooncogenes, particularly transcription factor protooncogenes, have important roles regulating proliferation and differentiation. The antisense methodologies produce inhibition of specific gene products by exploiting hybridization of complementary nucleic acids, resulting in decreased mRNA stability, or through a block in mRNA processing, transport, or translation. Transfection of antisense plasmids generally requires the isolation of stable transformants that can produce artifactual results due to biased selection pressures and direct effects of inducing agents on experimental systems. The addition of antisense oligonucleotides can occasionally generate artifactual results due to toxicity and not as a direct consequence of hybridization and consequent antisense inhibition. Major considerations for design of antisense oligonucleotide include: (1) site of target sequence within gene, (2) length of antisense oligonucleotide, and (3) secondary structure of antisense oligonucleotide.

The multiple isoforms of PDGF induce fibroblastic mitogenesis through two distinct PDGF receptors, alpha and beta. The molecular mechanisms by which these alpha and beta PDGF receptors regulate gene expression are poorly understood. We present data which indicates that differential induction of c-fos gene expression by PDGF isoforms occurs through distinct PDGF alpha and beta receptor-mediated signaling pathways. Comparison of PDGF-AA with PDGF-BB stimulation showed that PDGF-BB induced prolonged expression of the c-fos gene in BALB/c-3T3 cells, but that PDGF-AA induced more potent activation of the serum response element (SRE) in transient transfection assays. PDGF-AA, which binds alpha but not beta PDGF receptors, could only induce the SRE through a protein kinase C (PKC)-dependent pathway, whereas PDGF-BB, which binds both alpha and beta PDGF receptors, could also induce the SRE through a PKC-independent pathway. These results suggest that PDGF alpha receptors activate the PKC-dependent signaling pathway while PDGF beta receptors also activate a PKC-independent pathway. In addition, we found that PDGF-BB could induce another c-fos promoter element within the -90 to +10 region, suggesting that the more potent mitogenic effect and prolonged c-fos gene expression induced by PDGF-BB may result from cooperativity between more than one c-fos promoter elements.

Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGF beta and TNF alpha downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNF alpha produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 microM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGF beta 1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGF beta and TNF alpha, but that TNF alpha possesses an additional effect that is independent of c-myc expression.

Using an antisense RNA approach to eliminate endogenous expression of the c-fos protein, we have verified by nuclear run-on and transient expression assays that the Fos protein is a negative regulator of its own transcription in vivo. The negative autoregulation of the c-fos promoter by Fos was further confirmed by overexpression of an antisense-resistant c-fos expressing vector. Antisense mapping of the c-fos promoter demonstrated that the serum responsive element (SRE) represents the major site for c-fos suppression only during the first hour, but that additional adjacent DNA sequences are required for suppression at later times. We propose that antisense inhibition of transcriptional repressors provides a useful method for analyzing the significance and mechanism of transcriptional repression in vivo.

Two distinct regions of the transforming growth factor-beta 1 (TGF-beta 1) promoter are responsive to autoregulation and activation by phorbol ester (12-O-tetradecanoylphorbol-13-acetate): sequences located between nucleotides -454 to -323 (first promoter) and between the two transcriptional start sites. We have now characterized in detail the induction of the second promoter (sequences between nucleotides + 1 to +271) of the TGF-beta 1 gene by both TGF-beta 1 and phorbol ester. By assaying progressively deleted mutations in the second promoter, we have found two regions responsible for the induction; each contains a phorbol ester-responsive element. In vitro transcription of the second promoter-chloramphenicol acetyltransferase chimeric genes using nuclear extracts of A-549 cells showed that deletion of the putative phorbol ester-responsive elements results in a 70-80% decrease in activity. DNase I footprinting and gel mobility shift assays showed that binding to an Sp1 site and the putative TRE elements are required for maximal expression of the second promoter region of the TGF-beta 1 gene. These results suggest that AP-1, which is capable of conferring phorbol ester or TGF-beta 1 responsiveness, is the major transcription factor involved in the second promoter-derived transcription of the TGF-beta 1 gene.

The rapid induction of the proto-oncogene c-fos by growth factors and other bioactive agents, and the recent evidence that the c-fos protein (Fos) is associated with transcriptional complexes, suggests that Fos may represent an integral part of an intracellular messenger pathway that triggers changes in gene expression and ultimately phenotypic alterations. This report examines the role of c-fos in growth factor stimulation of transin, a matrix-degrading secreted metalloproteinase. Platelet-derived growth factor (PDGF) stimulation of transin RNA was blocked by a selective reduction in Fos synthesis with antisense c-fos mRNA, whereas epidermal growth factor (EGF) stimulation of transin occurred despite an equivalent inhibition of Fos levels. The stimulatory effect of both PDGF and EGF on transin transcription involved factors recognizing the sequence TGAGTCA, which is found in the transin promoter and is reported to be a binding site for the transcriptional factor Jun/AP-1 and for associated Fos and Fos-related complexes. Thus both Fos-dependent and Fos-independent pathways exist for growth factor regulation of gene expression, and both effects may be mediated through the same cis-acting transcription element.