Knockout model reveals the role of Nischarin in mammary gland development, breast tumorigenesis and response to metformin treatment.

Previously, our lab discovered the protein Nischarin and uncovered its role in regulating cell migration and invasion via its interactions with several proteins. We subsequently described a role for Nischarin in breast cancer, in which it is frequently underexpressed. To characterize Nischarin's role in breast tumorigenesis and mammary gland development more completely, we deleted a critical region of the Nisch gene (exons 7-10) from the mouse genome and observed the effects. Mammary glands in mutant animals showed delayed terminal end bud formation but did not develop breast tumors spontaneously. Therefore, we interbred the animals with transgenic mice expressing the mouse mammary tumor virus-polyoma middle T-antigen (MMTV-PyMT) oncogene. The MMTV-PyMT mammary glands lacking Nischarin showed increased hyperplasia compared to wild-type animal tissues. Furthermore, we observed significantly increased tumor growth and metastasis in Nischarin mutant animals. Surprisingly, Nischarin deletion decreased activity of AMPK and subsequently its downstream effectors. Given this finding, we treated these animals with metformin, which enhances AMPK activity. Here, we show for the first time, metformin activates AMPK signaling and inhibits tumor growth of Nischarin lacking PyMT tumors suggesting a potential use for metformin as a cancer therapeutic, particularly in the case of Nischarin-deficient breast cancers.

CRISPR/Cas9-mediated knockout of Lim-domain only four retards organ of Corti cell growth.

Lim-domain only 4 (LMO4) plays a critical role in mediating the ototoxic side-effects of cisplatin, a highly effective anti-cancer drug. However, the signaling mechanism by which cochlear LMO4 mediates otopathology is yet to be fully understood. Knockout cell culture models are useful tools for investigating the functional roles of novel genes and delineating associated signaling pathways. Therefore, LMO4 knockout organ of Corti cells were generated by using the CRISPR (clustered regularly interspersed short palindromic repeats)/Cas9 (CRISPR-associated protein 9) system. Successful knockout of LMO4 in UB/OC1 cells was verified by the absence of LMO4 protein bands in immunoblots. Though the Knockout of LMO4 retarded the growth rate and the migratory potential of the cells it did not inhibit their long-term viability as the LMO4 knockout UB/OC1 cells were able to survive, proliferate, and form colonies. In addition, the knockout of LMO4 did not alter the expression of myosin VIIa, a biomarker of hair cells, suggesting that the knockout cells retain important characteristic features of cochlear sensory receptor cells. Thus, the findings of this study indicate that CRISPR/Cas9 system is a simple and versatile method for knocking out genes of interest in organ of Corti cells and that LMO4 knockout UB/OC1 cells are viable experimental models for studying the functional role of LMO4 in ototoxicity.

Integrin subunits alpha5 and alpha6 regulate cell cycle by modulating the chk1 and Rb/E2F pathways to affect breast cancer metastasis.

BACKGROUND: Although integrins have been implicated in the progression of breast cancer, the exact mechanism whereby they exert this regulation is clearly not understood. To understand the role of integrins in breast cancer, we examined the expression levels of several integrins in mouse breast cancer cell lines by flow cytometry and the data were validated by Western and RT-PCR analysis. The importance of integrins in cell migration and cell invasion was examined by in vitro assays. Further the effect of integrins on metastasis was investigated by in vivo experimental metastasis assays using mouse models. RESULTS: Integrin α5 subunit is highly expressed in the nonmetastatic cell line 67NR and is significantly low in the highly invasive cell line 4T1. In contrast, expression levels of integrin α6 subunit are high in 4T1 cells and low in 67NR cells. In vitro data indicated that overexpression of α5 subunit and knockdown of α6 integrin subunit inhibited cell proliferation, migration, and invasion. Our in vivo findings indicated that overexpression of integrin α5 subunit and knockdown of α6 subunit decreased the pulmonary metastasis property of 4T1 cells. Our data also indicated that overexpression of alpha 5 integrin subunit and suppression of alpha6 integrin subunit inhibited cells entering into S phase by up-regulating p27, which results in downregulation of cyclinE/CDK2 complexes, This suggests that these integrins regulate cell growth through their effects on cell-cycle-regulated proteins. We also found that modulation of these integrins upregulates E2F, which may induce the expression of chk1 to regulate cdc25A/cyclin E/CDK2/Rb in a feedback loop mechanism. CONCLUSION: This study indicates that Integrin α5 subunit functions as a potential metastasis suppressor, while α6 subunit functions as a metastasis promoter. The modulation of integrins reduces cdc25 A, another possible mechanism for downregulation of CDK2. Taken together we demonstrate a link between integrins and the chk1-cdc25-cyclin E/CDK2-Rb pathway.

ST14 (suppression of tumorigenicity 14) gene is a target for miR-27b, and the inhibitory effect of ST14 on cell growth is independent of miR-27b regulation.

MicroRNAs are noncoding, endogenous small RNAs that regulate target genes by cleavage of the targeted mRNA or translational repression. We investigated the microRNAome using 2-color microarrays in a highly invasive human breast cancer cell line, MDA-MB-231 (subline 4175) and a noninvasive breast epithelial cell line, MCF10A. We found 13 microRNAs that were up-regulated, and nine were down-regulated significantly in 4175 cells (p < 0.05, -fold change >2) compared with MCF10A cells. Interestingly, miR-27b and its putative target gene, ST14 (suppressor of tumorigenicity 14), had inverse expression pattern in breast cancer cells. The 3'-untranslated region of ST14 contains a regulatory element for miR-27b, and our luciferase experiments indicate that antisense miR-27b enhances ST14 expression in cancer cells. Furthermore, antagomir of miR-27b suppressed cell invasion in 4175 cells, whereas pre-miR-27b stimulated invasion in moderately invasive ZR75 breast cancer cells. In addition, ST14 reduces cell proliferation as well as cell migration and invasion. Analysis of human breast tumors revealed that miR-27b expression increases during cancer progression, paralleling a decrease in ST14 expression. Furthermore, our data indicate that ST14 inhibits cells from entering into S phase by up-regulating p27, which results in down-regulation of cyclin E-CDK2 complexes, suggesting ST14 reduces cell growth through its effects on cell cycle-related proteins. Introduction of miR-27b into ST14-expressing cells did not suppress the effect on cell growth. These findings suggest that ST14 plays an important role in several biological processes, and some effects are not completely dependent on miR-27b regulation.

Exosomes from Nischarin-Expressing Cells Reduce Breast Cancer Cell Motility and Tumor Growth.

Exosomes are small extracellular microvesicles that are secreted by cells when intracellular multivesicular bodies fuse with the plasma membrane. We have previously demonstrated that Nischarin inhibits focal adhesion formation, cell migration, and invasion, leading to reduced activation of focal adhesion kinase. In this study, we propose that the tumor suppressor Nischarin regulates the release of exosomes. When cocultured on exosomes from Nischarin-positive cells, breast cancer cells exhibited reduced survival, migration, adhesion, and spreading. The same cocultures formed xenograft tumors of significantly reduced volume following injection into mice. Exosomes secreted by Nischarin-expressing tumors inhibited tumor growth. Expression of only one allele of Nischarin increased secretion of exosomes, and Rab14 activity modulated exosome secretions and cell growth. Taken together, this study reveals a novel role for Nischarin in preventing cancer cell motility, which contributes to our understanding of exosome biology. SIGNIFICANCE: Regulation of Nischarin-mediated exosome secretion by Rab14 seems to play an important role in controlling tumor growth and migration.See related commentary by McAndrews and Kalluri, p. 2099.

Targeting nitrative stress for attenuating cisplatin-induced downregulation of cochlear LIM domain only 4 and ototoxicity.

Cisplatin-induced ototoxicity remains a primary dose-limiting adverse effect of this highly effective anticancer drug. The clinical utility of cisplatin could be enhanced if the signaling pathways that regulate the toxic side-effects are delineated. In previous studies, we reported cisplatin-induced nitration of cochlear proteins and provided the first evidence for nitration and downregulation of cochlear LIM domain only 4 (LMO4) in cisplatin ototoxicity. Here, we extend these findings to define the critical role of nitrative stress in cisplatin-induced downregulation of LMO4 and its consequent ototoxic effects in UBOC1 cell cultures derived from sensory epithelial cells of the inner ear and in CBA/J mice. Cisplatin treatment increased the levels of nitrotyrosine and active caspase 3 in UBOC1 cells, which was detected by immunocytochemical and flow cytometry analysis, respectively. The cisplatin-induced nitrative stress and apoptosis were attenuated by co-treatment with SRI110, a peroxynitrite decomposition catalyst (PNDC), which also attenuated the cisplatin-induced downregulation of LMO4 in a dose-dependent manner. Furthermore, transient overexpression of LMO4 in UBOC1 cells prevented cisplatin-induced cytotoxicity while repression of LMO4 exacerbated cisplatin-induced cell death, indicating a direct link between LMO4 protein levels and cisplatin ototoxicity. Finally, auditory brainstem responses (ABR) recorded from CBA/J mice indicated that co-treatment with SRI110 mitigated cisplatin-induced hearing loss. Together, these results suggest that cisplatin-induced nitrative stress leads to a decrease in the levels of LMO4, downregulation of LMO4 is a critical determinant in cisplatin-induced ototoxicity, and targeting peroxynitrite could be a promising strategy for mitigating cisplatin-induced hearing loss.

Cisplatin-induced apoptosis in auditory, renal, and neuronal cells is associated with nitration and downregulation of LMO4.

Cytotoxic effects of cisplatin occur primarily through apoptosis. Though several pro- and anti-apoptotic signaling molecules have been identified to play an important role in mediating the ototoxic, nephrotoxic, and neurotoxic side-effects of cisplatin, the underlying mechanism is yet to be fully characterized. We reported that nitration of LIM domain only 4 (LMO4), a transcriptional regulator, facilitates cochlear apoptosis in cisplatin-induced ototoxicity. However, its role in cisplatin-mediated nephrotoxicity and neurotoxicity is poorly understood. Therefore, HK2, and SH-SY5Y cells were employed along with UBOC1 cells, to investigate the perturbations of LMO4 in cisplatin-induced cytotoxicity, in renal, neuronal, and auditory cells, respectively. Cisplatin induced an increase in the expression of active caspase-3, indicating cellular apoptosis, and increased the nitration of proteins, 24 h post-treatment. Immunostaining with anti-nitrotyrosine and anti-LMO4 indicated that nitrotyrosine co-localized with LMO4 protein in cisplatin treated cells. Immunoblotting with anti-LMO4 indicated that cisplatin induced a decrease in LMO4 protein levels. However, a corresponding decrease in LMO4 gene levels was not observed. Inhibition of protein nitration with SRI110, a peroxynitrite decomposition catalyst, attenuated cisplatin-induced downregulation of LMO4. More importantly, overexpression of LMO4 mitigated the cytotoxic effects of cisplatin in UBOC1 cells while a dose-dependent decrease in LMO4 protein strongly correlated with cell viability in UBOC1, HK2, and SH-SY5Y cells. Collectively, these findings suggested a potential role of LMO4 in facilitating the cytotoxic effects of cisplatin in auditory, renal, and neuronal cells.

Role of Rho GTPases and their regulators in cancer progression.

Rho family of GTPases is an ubiquitiously expressed and evolutionarily conserved family of GTP binding proteins that regulate actin dynamics and intracellular signaling. Among the Rho family GTPases, three members RhoA, Rac1 and CDC42 have been well characterized. They each play pivotal roles in gene expression, cell proliferation, apoptosis and various cellular functions. They are driven by signaling from RhoGDIs, RhoGEFs, RhoGAPs and cell surface receptors. Abnormalities in Rho GTPase function have major consequences on cell behavior. Over expression of Rho GTPases is associated with reorganization of actin cytoskeleton, an increase in cell migration, invasion and metastasis which are important aspects of cancer progression. This review will explore these Rho GTPases and the function of their associated signaling pathways in different types of cancers.

Molecular characterization of the tumor-suppressive function of nischarin in breast cancer.

BACKGROUND: Nischarin (encoded by NISCH), an α5 integrin-binding protein, has been identified as a regulator of breast cancer cell invasion. We hypothesized that it might be a tumor suppressor and were interested in its regulation. METHODS: We examined nischarin expression in approximately 300 human breast cancer and normal tissues using quantitative polymerase chain reaction and immunohistochemistry. Loss of heterozygosity analysis was performed by examining three microsatellite markers located near the NISCH locus in normal and tumor tissues. We generated derivatives of MDA-MB-231 human metastatic breast cancer cells that overexpressed nischarin and measured tumor growth from these cells as xenografts in mice; metastasis by these cells after tail vein injection; and α5 integrin expression, Rac, and focal adhesion kinase (FAK) signaling using western blotting. We also generated clones of MCF-7 human breast cancer cells in which nischarin expression was silenced and measured tumor growth in mouse xenograft models (n = 5 for all mouse experiments). P values were from two-sided Student t tests in pairwise comparisons. RESULTS: Normal human breast tissue samples had statistically significantly higher expression of nischarin mRNA compared with tumor tissue samples (mean level in normal breast = 50.7 [arbitrary units], in breast tumor = 16.49 [arbitrary units], difference = 34.21, 95% confidence interval [CI] = 11.63 to 56.79, P = .003), and loss of heterozygosity was associated with loss of nischarin expression. MDA-MB-231 cells in which nischarin was overexpressed had statistically significantly reduced tumor growth and metastasis compared with parental MDA-MB-231 cells (mean volume at day 40, control vs nischarin-expressing tumors, 1977 vs 42.27 mm(3), difference = 1935 mm(3), 95% CI = 395 to 3475 mm(3), P = .025). Moreover, MCF-7 tumor xenografts in which nischarin expression was silenced grew statistically significantly faster than parental cells (mean volume at day 63, tumors with scrambled short hairpin RNA [shRNA] vs with nischarin shRNA, 224 vs 1262 mm(3), difference = 1038 mm(3), 95% CI = 899.6 to 1176 mm(3), P < .001). Overexpression of nischarin was associated with decreased α5 integrin expression, FAK phosphorylation, and Rac activation. CONCLUSION: Nischarin may be a novel tumor suppressor that limits breast cancer progression by regulating α5 integrin expression and subsequently α5 integrin-, FAK-, and Rac-mediated signaling.