The hippocampus, a major site of neurogenesis in the adult brain, plays an important role in memory. Based on earlier observations where exposure to high-intensity noise not only caused hearing loss but also impaired memory function, it is conceivably that noise exposure may suppress hippocampal neurogenesis. To evaluate this possibility, nine rats were unilaterally exposed for 2 h to a high-intensity, narrow band of noise centered at 12 kHz at 126 dB SPL. The rats were also screened for noise-induced tinnitus, a potential stressor which may suppress neurogenesis. Five rats developed persistent tinnitus-like behavior while the other four rats showed no signs of tinnitus. Age-matched sham controls showed no signs of hearing loss or tinnitus. The inner ear and hippocampus were evaluated for sensory hair cell loss and neurogenesis 10 weeks post-exposure. All noise exposed rats showed severe loss of sensory hair cells in the noise-exposed ear, but essentially no damage in the unexposed ear. Frontal sections from the hippocampus were immunolabeled for doublecortin to identify neuronal precursor cells, or Ki67 to label proliferating cells. Noise-exposed rats showed a significant reduction of neuronal precursors and fewer dividing cells as compared to sham controls. However, we could not detect any difference between rats with behavioral evidence of tinnitus versus rats without tinnitus. These results show for the first time that high intensity noise exposure not only damages the cochlea but also causes a significant and persistent decrease in hippocampal neurogenesis that may contribute to functional deficits in memory.

Efficient therapeutics and early detection has helped to increase breast cancer survival rates over the years. However, the recurrence of breast cancer remains to be a problem and this may be due to the presence of a small population of cells, called tumor initiating cells (TICs). Breast TICs are resistant to drugs, difficult to detect, and exhibit high self-renewal capabilities. In this study, layer by layer (LBL) small interfering RNA (siRNA) nanovectors (SNVs) were designed to target breast TICs. SNVs were fabricated using alternating layers of poly-L-lysine and siRNA molecules on gold (Au) nanoparticle (NP) surfaces. The stability, cell uptake, and release profile for SNVs were examined. In addition, SNVs reduced TIC-related STAT3 expression levels, CD44+/CD24−/EpCAM+ surface marker levels and the number of mammospheres formed compared to the standard transfection agent. The data from this study show, for the first time, that SNVs in LBL assembly effectively delivers STAT3 siRNA and inhibit the growth of breast TICs in vitro.

A method to purify enzymes utilizing their specific biological affinity and catalytic specificity is described. For this chromatographic technique, an enzyme binds immobilized substrate coupled to a column in the absence of a cofactor required for catalysis but permissive for substrate binding. After washing, the missing cofactor is added to the column mobile phase, and the enzyme converts substrate into product and elutes from the column. A single-step purification of EcoRI endonuclease using a sequence-specific DNA column (containing the GAATTC motif coupled to cyanogen bromide-activated Sepharose 4B) binds EcoRI in the absence of Mg2+ and elutes when Mg2+ is applied in a highly purified state. Although the method described is specific for EcoRI, it can be readily modified for the purification of DNA polymerases and other enzymes. Furthermore, many of the same materials are also used for transcription factor purification. This protocol can be completed within 4-6 d.

Triple negative breast cancer (TNBC) is the most aggressive and lethal form of cancer characterized by lack of estrogen, progesterone and Her2 receptors. It is prevalent in women of African American descent, often present in younger and premenopausal women. It shows high risk of recurrence and frequently metastasizes to lungs and brain, resulting in poor overall prognosis. There is no targeted therapy for TNBC as it does not respond to hormonal therapy and is intrinsically resistance to conventional chemotherapy. It is therefore imperative to understand the mechanism of survival of these cancers and unravel its biological pathways and modes of progression. We have previously described breast cancer stem cells (BCSC) to be intrinsically resistant to treatment which is further confirmed by recent publications by other groups that describe direct functional evidence for the same. Using genomic assays, we traced a BCSC gene signature comprising of 477 genes derived from patient biopsies. On selective shRNA knockdown of these genes we identified RPL39 and MLF2 as the top two candidates that affect BCSC self-renewal. Selective siRNA knockdown of RPL39 and MLF2 in human cancer xenografts, showed reduced tumor volume and lung metastases with a concomitant decrease in BCSC markers. Thus, targeting BCSCs in combination with chemotherapy should eliminate the heterogeneous populations within a tumor. Additionally, next generation RNA-seq confirmed mutations in RPL39 and MLF2 in 50% of lung metastases from breast cancer patients. In vitro and in vivo siRNA knockdown of RPL39 and MLF2 showed decrease in nitric oxide synthase, suggesting that these genes are driven by nitric oxide signaling. In conclusion this study reveals novel tumor initiating genes, RPL39 and MLF2 that target the breast cancer stem cells and also show impact on lung metastasis. Our findings enhance the understanding of treatment resistant breast cancer stem cells, the mutations that cause metastases and also lay foundation for developing new therapies for such cancers with poor prognosis. Citation Format: Bhuvanesh Dave, Sergio Granados, Junhua Mai, Dong Soon Choi, Ding Cheng Gao, Sucharita Mitra, Haifa Shen, Senthil Muthuswamy, Vivek Mittal, Mauro Ferrari, Jenny Chang. Identification of tumor initiating genes RPL39 and MLF2 that mediate lung metastasis through nitric oxide signaling and mesenchymal to epithelial transition. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2712. doi:10.1158/1538-7445.AM2013-2712

Autophagy is an evolutionarily conserved lysosomal pathway for degradation of cytoplasmic proteins, macromolecules, and organelles. It is defined as a cell survival pathway which allows cells to survive nutrient and environmental stress. Tumor initiating cells (TICs) survive radiation, endocrine therapy, and chemotherapy, making them the primary cause of relapse in breast cancer patients. Our previous studies have identified autophagy as the mechanism of survival in these treatment-resistant populations in letrozole- as well as chemotherapy-treated patient samples. In this study we confirmed our previous finding by using a real time PCR low density array of 84 genes related to autophagy in 9 paired pre- vs. post-letrozole treated clinical samples, where 60 out of 84 genes demonstrated a statically significant change (p<0.05). Autophagy inhibitors like N-acetyl cysteine and catalase demonstrate a significant reduction in TICs using mammosphere forming efficiency (MSFE) (p<0.05) and flow cytometry of CD44+/CD24- (p<0.05) cells in three triple negative breast cancer cell lines (MDA-MB231, SUM159 and BT549). Recent evidence from our group had determined a role for hypoxia and reactive nitrogen species in TIC inhibition. Here we demonstrate that both catalase and N-acetyl cysteine inhibit TICs by a mechanism that significantly reduces inducible nitric oxide synthase and HIF-1α, implicating both hypoxia and reactive nitrogen species in the process. We will be confirming our finding in-vivo using patient-derived and cell line human xenografts and further examine the cross talk between hypoxia and reactive nitrogen species as it relates to autophagy. The understanding of this process may allow us to target TICs in a novel mechanistic way. Citation Format: Bhuvanesh Dave, Sucharita Mitra. Autophagy: a mechanism of resistance to therapy in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3768. doi:10.1158/1538-7445.AM2013-3768