BRCA1 Deficiency Exacerbates Estrogen-Induced DNA Damage and Genomic Instability

Germline mutations in BRCA1 predispose carriers to a high incidence of breast and ovarian cancers. BRCA1 functions to maintain genomic stability through critical roles in DNA repair, cell-cycle arrest, and transcriptional control. A major question has been why BRCA1 loss or mutation leads to tumors mainly in estrogen-regulated tissues, given that BRCA1 has essential functions in all cell types. Here, we report that estrogen and estrogen metabolites can cause DNA double-strand breaks (DSB) in estrogen receptora- negative breast cells and that BRCA1 is required to repair these DSBs to prevent metabolite-induced genomic instability.We found that BRCA1 also regulates estrogen metabolism and metabolite-mediated DNA damage by repressing the transcription of estrogen-metabolizing enzymes, such as CYP1A1, in breast cells. Finally, we used a knock-in human cell model with a heterozygous BRCA1 pathogenic mutation to show how BRCA1 haploinsufficiency affects these processes. Our findings provide pivotal new insights into why BRCA1 mutation drives the formation of tumors in estrogen-regulated tissues, despite the general role of BRCA1 in DNA repair in all cell types. © 2014 American Association for Cancer Research.

Deficiency of the ferrous iron transporter FeoAB is linked with metronidazole resistance in Bacteroides fragilis

Background Metronidazole is the most commonly used antimicrobial for Bacteroides fragilis infections and is recommended for prophylaxis of colorectal surgery. Metronidazole resistance is increasing and the mechanisms of resistance are not clear.

Methods A transposon mutant library was generated in B. fragilis 638R (BF638R) to identify the genetic loci associated with resistance to metronidazole.

Results Thirty-two independently isolated metronidazole-resistant mutants had a transposon insertion in BF638R_1421 that encodes the ferrous transport fusion protein (feoAB). Deletion of feoAB resulted in a 10-fold increased MIC of metronidazole for the strain. The metronidazole MIC for the feoAB mutant was similar to that for the parent strain when grown on media supplemented with excess iron, suggesting that the increase seen in the MIC of metronidazole was due to reduced cellular iron transport in the feoAB mutant. The furA gene repressed feoAB transcription in an iron-dependent manner and disruption of furA resulted in constitutive transcription of feoAB, regardless of whether or not iron was present. However, disruption of feoAB also diminished the capacity of BF638R to grow in a mouse intraperitoneal abscess model, suggesting that inorganic ferrous iron assimilation is essential for B. fragilis survival in vivo.

Conclusions Selection for feoAB mutations as a result of metronidazole treatment will disable the pathogenic potential of B. fragilis and could contribute to the clinical efficacy of metronidazole. While mutations in feoAB are probably not a direct cause of clinical resistance, this study provides a key insight into intracellular metronidazole activity and the link with intracellular iron homeostasis.

Sex hormone binding globulin and insulin resistance

Sex hormone binding globulin (SHBG) is a glycoprotein composed of two 373-amino-acid subunits. The SHBG gene and a promotor region have been identified. The SHBG receptor has yet to be cloned but is known to act through a G-protein-linked second-messenger system following plasma membrane binding. The principal function of SHBG has traditionally been considered to be that of a transport protein for sex steroids, regulating circulating concentrations of free (unbound) hormones and their transport to target tissues. Recent research suggests that SHBG has functions in addition to the binding and transport of sex steroids. Observational studies have associated a low SHBG concentration with an increased incidence of type 2 diabetes mellitus (DM) independent of sex hormone levels in men and women. Genetic studies using Mendelian randomization analysis linking three single nucleotide polymorphisms of the SHBG gene to risk of developing type 2 DM suggest SHBG may have a role in the pathogenesis of type 2 DM. The correlation between SHBG and insulin resistance that is evident in a number of cross-sectional studies is in keeping with the suggestion that the association between SHBG and incidence of type 2 DM is explained by insulin resistance. Several potential mechanisms may account for this association, including the identification of dietary factors that influence SHBG gene transcription. Further research to characterize the SHBG-receptor and the SHBG second messenger system is required. An interventional study examining the effects on insulin resistance of altering SHBG concentrations may help in determining whether this association is causal.

Conserved histidine of metal transporter AtNRAMP1 is crucial for optimal plant growth under manganese deficiency at chilling temperatures

Manganese (Mn) is an essential nutrient required for plant growth, in particular in the process of photosynthesis. Plant performance is influenced by various environmental stresses including contrasting temperatures, light or nutrient deficiencies. The molecular responses of plants exposed to such stress factors in combination are largely unknown. 

Screening of 108 Arabidopsis thaliana (Arabidopsis) accessions for reduced photosynthetic performance at chilling temperatures was performed and one accession (Hog) was isolated. Using genetic and molecular approaches, the molecular basis of this particular response to temperature (GxE interaction) was identified. 

Hog showed an induction of a severe leaf chlorosis and impaired growth after transfer to lower temperatures. We demonstrated that this response was dependent on the nutrient content of the soil. Genetic mapping and complementation identified NRAMP1 as the causal gene. Chlorotic phenotype was associated with a histidine to tyrosine (H239Y) substitution in the allele of Hog NRAMP1. This led to lethality when Hog seedlings were directly grown at 4 degrees C. 

Chemical complementation and hydroponic culture experiments showed that Mn deficiency was the major cause of this GxE interaction. For the first time, the NRAMP-specific highly conserved histidine was shown to be crucial for plant performance.

Mitotic Arrest Deficiency Protein 2 (MAD2) and Histone Deacetylase 6 (HDAC6) Present a Complex Relationship in Their Regulation and Expression and Subsequent Impact on Chemoresponsiveness

Introduction:
Ovarian cancer patients presenting with advanced stage (III/IV)
canceraretreatedwithcarboplatinumincombinationwithpaclitaxel.Despitea
significant initial response rate, fewer than 20% of patients become long-term
survivors. We have published that low MAD2 expression levels associate with
reduced progression free survival (PFS) in patients with high-grade serous
epithelial ovarian cancer (EOC). Moreover, we have demonstrated that MAD2
expressionisdown-regulatedbythemicroRNAmiR-433(
Furlong et al., 2011
).
Interestingly, miR-433 also down-regulates HDAC6 (
Simon et al., 2010
), which
uniquely deacetylates
a
-tubulin prior to HDAC6s binding to
b
-tubulin.
In vitro
studies have shown that HDAC6 inhibition in combination with paclitaxel
treatment enhances chemoresistant cancer cell death. To date, an interaction
between MAD2 and HDAC6 has not been reported.
Experimental design:
MAD2 and HDAC6 immunohistochemistry (IHC) and
Western blot analyses were performed to investigate the role of HDAC6 and
MAD2 in chemoresistance to paclitaxel in high-grade serous EOC.
Results and Discussion:
In vitro
experiments demonstrated that overex-
pression of pre-miR-433, which targets MAD2, resulted in down-regulation
of HDAC6 in EOC cell lines. High levels of HDAC6 are co-expressed with
MAD2 in the paclitaxel resistant UPN251 and OVCAR7 cell lines. While, all
4 paclitaxel resistant EOC cell lines express higher levels of miR-433 than
the paclitaxel sensitive A2780 cells, only ovca432 and ovca433 demonstrated
down-regulation of both HDAC6 and MAD2. Paclitaxel binds to
b
-tubulin and
causesmicrotubulepolymerizationinpaclitaxelsensitivecellsasdemonstrated
by tubulin acetylation in A2780 cells. However, paclitaxel failed to cause a
significant acetylation of
a
-tubulin and microtubule stabilisation in the resistant
UPN251 cells. Therefore resistance in this cell line may be mediated by
aberrantly high HDAC6 activity. We have previously shown that MAD2 knock-
down cells are resistant to paclitaxel (
Furlong F., et al., 2011; Prencipe M.,
et al., 2009
). We measured HDAC6 protein expression in MAD2 knockdown
cells and showed that MAD2 knockdown is associated with concomitant
up-regulation of HDAC6. We hypothesise that the up-regulation of HDAC6
by MAD2 knockdown renders cancer cells more resistant to paclitaxel and
increases the invasive potential of these cells. On-going experiments will test
this hypothesis. Lastly we have observed differential MAD2 and HDAC6 IHC
staining intensity in formalin fixed paraffin embedded EOC samples.
In conclusion
, we have reported on a novel interaction between MAD2 and
HDAC6 which may have important consequences for paclitaxel resistant EOC.
Moreover, understanding chemo-responsiveness in ovarian tumours will lead
to improved patient management and treatment options for women diagnosed
with this disease