Laura E. Hays

BRCAness profile of sporadic ovarian cancer predicts disease recurrence.

Background The consequences of defective homologous recombination (HR) are not understood in sporadic ovarian cancer, nor have the potential role of HR proteins other than BRCA1 and BRCA2 been clearly defined. However, it is clear that defects in HR and other DNA repair pathways are important to the effectiveness of current therapies. We hypothesize that a subset of sporadic ovarian carcinomas may harbor anomalies in HR pathways, and that a BRCAness profile (defects in HR or other DNA repair pathways) could influence response rate and survival after treatment with platinum drugs. Clinical availability of a BRCAness profile in patients and/or tumors should improve treatment outcomes. Objective To define the BRCAness profile of sporadic ovarian carcinoma and determine whether BRCA1, PARP, FANCD2, PTEN, H2AX, ATM, and P53 protein expression correlates with response to treatment, disease recurrence, and recurrence-free survival. Materials and Methods Protein microarray analysis of ovarian cancer tissue was used to determine protein expression levels for defined DNA repair proteins. Correlation with clinical and pathologic parameters in 186 patients with advanced stage III–IV and grade 3 ovarian cancer was analyzed using Chi square, Kaplan-Meier method, Cox proportional hazard model, and cumulative incidence function. Results High PARP, FANCD2 and BRCA1 expressions were significantly correlated with each other; however, elevated p53 expression was associated only with high PARP and FANCD2. Of all patients, 9% recurred within the first year. Among early recurring patients, 41% had high levels of PARP, FANCD2 and P53, compared to 19.5% of patients without early recurrence (p = 0.04). Women with high levels of PARP, FANCD2 and/or P53 had first year cumulative cancer incidence of 17% compared with 7% for the other groups (P = 0.03). Conclusions Patients with concomitantly high levels of PARP, FANCD2 and P53 protein expression are at increased risk of early ovarian cancer recurrence and platinum resistance.

Cytogenetic Instability in Ovarian Epithelial Cells from Women at Risk of Ovarian Cancer

Fanconi anemia is an inherited cancer predisposition disease characterized by cytogenetic and cellular hypersensitivity to cross-linking agents. Seeking evidence of Fanconi anemia protein dysfunction in women at risk of ovarian cancer, we screened ovarian surface epithelial cells from 25 primary cultures established from 22 patients using cross-linker hypersensitivity assays. Samples were obtained from (a) women at high risk for ovarian cancer with histologically normal ovaries, (b) ovarian cancer patients, and (c) a control group with no family history of breast or ovarian cancer. In chromosomal breakage assays, all control cells were mitomycin C (MMC) resistant, but eight samples (five of the six high-risk and three of the eight ovarian cancer) were hypersensitive. Lymphocytes from all eight patients were MMC resistant. Only one of the eight patients had a BRCA1 germ-line mutation and none had BRCA2 mutations, but FANCD2 was reduced in five of the eight. Ectopic expression of normal FANCD2 cDNA increased FANCD2 protein and induced MMC resistance in both hypersensitive lines tested. No FANCD2 coding region or promoter mutations were found, and there was no genomic loss or promoter methylation in any Fanconi anemia genes. Therefore, in high-risk women with no BRCA1 or BRCA2 mutations, tissue-restricted hypersensitivity to cross-linking agents is a frequent finding, and chromosomal breakage responses to MMC may be a sensitive screening strategy because cytogenetic instability identified in this way antedates the onset of carcinoma. Inherited mutations that result in tissue-specific FANCD2 gene suppression may represent a cause of familial ovarian cancer.

p38 MAPK inhibition suppresses the TLR-hypersensitive phenotype in FANCC- and FANCA-deficient mononuclear phagocytes

Fanconi anemia, complementation group C (FANCC)-deficient hematopoietic stem and progenitor cells are hypersensitive to a variety of inhibitory cytokines, one of which, TNFα, can induce BM failure and clonal evolution in Fancc-deficient mice. FANCC-deficient macrophages are also hypersensitive to TLR activation and produce TNFα in an unrestrained fashion. Reasoning that suppression of inhibitory cytokine production might enhance hematopoiesis, we screened small molecules using TLR agonist-stimulated FANCC- and Fanconi anemia, complementation group A (FANCA)-deficient macrophages containing an NF-κB/AP-1-responsive reporter gene (SEAP). Of the 75 small molecules screened, the p38 MAPK inhibitor BIRB 796 and dasatinib potently suppressed TLR8-dependent expression of the reporter gene. Fanconi anemia (FA) macrophages were hypersensitive to the TLR7/8 activator R848, overproducing SEAP and TNFα in response to all doses of the agonist. Low doses (50nM) of both agents inhibited p38 MAPK-dependent activation of MAPKAPK2 (MK2) and suppressed MK2-dependent TNFα production without substantially influencing TNFα gene transcription. Overproduction of TNFα by primary FA cells was likewise suppressed by these agents and involved inhibition of MK2 activation. Because MK2 is also known to influence production and/or sensitivity to 2 other suppressive factors (MIP-1α and IFNγ) to which FA hematopoietic progenitor cells are uniquely vulnerable, targeting of p38 MAPK in FA hematopoietic cells is a rational objective for preclinical evaluation.

FANCA and FANCC modulate TLR and p38 MAPK dependent expression of IL-1β in macrophages

Hematopoietic stem and progenitor cells with inactivated Fanconi anemia (FA) genes, FANCA and FANCC, are hypersensitive to inflammatory cytokines. One of these, tumor necrosis factor α (TNF-α), is also overproduced by FA mononuclear phagocytes in response to certain Toll-like receptor (TLR) agonists, creating an autoinhibitory loop that may contribute to the pathogenesis of progressive bone marrow (BM) failure and selection of TNF-α-resistant leukemic stem cell clones. In macrophages, the TNF-α overproduction phenotype depends on p38 mitogen-activated protein kinase (MAPK), an enzyme also known to induce expression of other inflammatory cytokines, including interleukin 1β (IL-1β). Reasoning that IL-1β might be involved in a like autoinhibitory loop, we determined that (1) TLR activation of FANCA- and FANCC-deficient macrophages induced overproduction of both TNF-α and IL-1β in a p38-dependent manner; (2) exposure of Fancc-deficient BM progenitors to IL-1β potently suppressed the expansion of multipotent progenitor cells in vitro; and (3) although TNF-α overexpression in FA cells is controlled posttranscriptionally by the p38 substrate MAPKAPK-2, p38-dependent overproduction of IL-1β is controlled transcriptionally. We suggest that multiple inflammatory cytokines overproduced by FANCA- and FANCC-deficient mononuclear phagocytes may contribute to the progressive BM failure that characterizes FA, and that to achieve suppression of this proinflammatory state, p38 is a more promising molecular therapeutic target than either IL-1β or TNF-α alone.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Human FANCC is hypomorphic in murine Fancc-deficient cells

Fancc suppresses cross-linker-induced genotoxicity, modulates growth-inhibitory cytokine responses, and modulates endotoxin responses. Although loss of the latter function is known to account for endotoxin-induced marrow failure in murine Fancc (mFancc)-deficient mice, some argue that cytokine and endotoxin hypersensitivities devolve simply from genomic instability. Seeking to resolve this question, we planned to ectopically express instructive human FANCC (hFANCC) mutants in murine Fancc-deficient hematopoietic stem cells. To first assure that hFANCC cDNA was competent in murine cells, we compared hFANCC and mFancc in complementation assays for cross-linking agent hypersensitivity and endotoxin hypersensitivity. We found that mFancc complemented murine Fancc-deficient cells in both assays, but that hFANCC fully suppressed only endotoxin hypersensitivity, not cross-linking agent hypersensitivity. These results support the notions that Fancc is multifunctional and that structural prerequisites for its genoprotective functions differ from those required to constrain endotoxin responses known to lead to marrow failure in Fancc-deficient mice.

Cytokine overproduction and crosslinker hypersensitivity are unlinked in Fanconi anemia macrophages

The Fanconi anemia proteins participate in a canonical pathway that repairs cross‐linking agent‐induced DNA damage. Cells with inactivated Fanconi anemia genes are universally hypersensitive to such agents. Fanconi anemia‐deficient hematopoietic stem cells are also hypersensitive to inflammatory cytokines, and, as importantly, Fanconi anemia macrophages overproduce such cytokines in response to TLR4 and TLR7/8 agonists. We questioned whether TLR‐induced DNA damage is the primary cause of aberrantly regulated cytokine production in Fanconi anemia macrophages by quantifying TLR agonist‐induced TNF‐α production, DNA strand breaks, crosslinker‐induced chromosomal breakage, and Fanconi anemia core complex function in Fanconi anemia complementation group C‐deficient human and murine macrophages. Although both M1 and M2 polarized Fanconi anemia cells were predictably hypersensitive to mitomycin C, only M1 macrophages overproduced TNF‐α in response to TLR‐activating signals. DNA damaging agents alone did not induce TNF‐α production in the absence of TLR agonists in wild‐type or Fanconi anemia macrophages, and mitomycin C did not enhance TLR responses in either normal or Fanconi anemia cells. TLR4 and TLR7/8 activation induced cytokine overproduction in Fanconi anemia macrophages. Also, although TLR4 activation was associated with induced double strand breaks, TLR7/8 activation was not. That DNA strand breaks and chromosome breaks are neither necessary nor sufficient to account for the overproduction of inflammatory cytokines by Fanconi anemia cells suggests that noncanonical anti‐inflammatory functions of Fanconi anemia complementation group C contribute to the aberrant macrophage phenotype and suggests that suppression of macrophage/TLR hyperreactivity might prevent cytokine‐induced stem cell attrition in Fanconi anemia.

Multifunctionality of the FA pathway

In this issue of Blood , Kim and colleagues demonstrate that the Fanconi anemia protein FANCP/SLX4 is multifunctional by identifying molecular domains that uniquely effect repair of DNA damage inflicted by either DNA cross-linkers or topoisomerase and PARP inhibitors.[1][1] Fanconi anemia (FA) is

A mismatched life in the stem cell pool

In this issue of Blood , Kenyon and colleagues demonstrate that age-related MLH1 hypermethylation leads to DNA mismatch repair defects and microsatellite instability in hematopoietic stem and progenitor cells.[1][1] The relative risks for leukemia, myelodysplastic syndrome, and some lymphoid

Abstract LB-157: A murine model of Fanconi anemia-associated head and neck cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Fanconi anemia (FA) predisposes patients to aggressive head-and-neck-squamous-cell-carcinomas (HNSCCs) early in life. These tumors are particularly difficult to treat because of the underlying hypersensitivity of all somatic cells from these patients to cross-linking agents. An in vivo model is thus badly needed to delineate the unique molecular mechanisms involved in tumor formation and to identify opportunities to exploit this mechanism therapeutically. Although mice with inactivating mutations of FA genes have been developed, these mice do not spontaneously develop HNSCCs. Therefore, we sought to develop our own model by treating wild-type (WT) and Fancc-deficient (FA-C) mice with the carcinogen 4-nitroquinoline 1-oxide for 45 weeks. Although tumor incidence, histological type and grade, and anatomical location were similar in WT and FA-C mice, cells isolated from FA-C tumors displayed higher levels of endogenous chromosomal instability compared to cells from WT tumors. None of the mice developed metastases, most likely because they required early euthanasia as tumor formation inhibited the ability to eat and drink. To compare the metastatic potential of WT and FA-C HNSCC cells, we subcutaneously injected these cells lines into the flanks of immunocompetent (C57Bl/6J) and immunodeficient (NOD/SCID) mice. Interestingly, tumors only formed in NOD/SCID mice indicating immune-mediated suppression of tumor growth in the immunocompetent mice. In NOD/SCID mice, 4 out of 6 WT and 3 out of 4 FA-C cell lines induced tumor formation. However, all of the tumor-forming FA-C lines produced aggressive tumors that penetrated into the retro-peritoneal space and/or produced hind-leg paralysis, while only 1 out of 4 WT lines invaded to this extent (a phenomenon that occurred in fewer than 25% of mice injected with this line). In addition, we found that tumor forming ability of FA-C HNSCC cell lines did not require loss of Fancc because FA-C lines transduced with wild-type Fancc remained fully tumorigenic in vivo. In conclusion, we have developed a model of FA related HNSCCs utilizing FA and WT cell lines. The model permits the outgrowth of tumors that are histologically, genomically, and biologically similar to corresponding tumors in humans. Currently we are utilizing this model to test the efficacy of small molecules in inhibiting FA tumor progression and are testing the role of the tumor microenvironment and the immune system in modulating the development of FA-related HNSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-157.