siRNA-Based Targeting of Cyclin E Overexpression Inhibits Breast Cancer Cell Growth and Suppresses Tumor Development in Breast Cancer Mouse Model

Cyclin E is aberrantly expressed in many types of cancer including breast cancer. High levels of the full length as well as the low molecular weight isoforms of cyclin E are associated with poor prognosis of breast cancer patients. Notably, cyclin E overexpression is also correlated with triple-negative basal-like breast cancers, which lack specific therapeutic targets. In this study, we used siRNA to target cyclin E overexpression and assessed its ability to suppress breast cancer growth in nude mice. Our results revealed that cyclin E siRNA could effectively inhibit overexpression of both full length and low molecular weight isoforms of cyclin E. We found that depletion of cyclin E promoted apoptosis of cyclin E-overexpressing cells and blocked their proliferation and transformation phenotypes. Significantly, we further demonstrated that administration of cyclin E siRNA could inhibit breast tumor growth in nude mice. In addition, we found that cyclin E siRNA synergistically enhanced the cell killing effects of doxorubicin in cell culture and this combination greatly suppressed the tumor growth in mice. In conclusion, our results indicate that cyclin E, which is overexpressed in 30% of breast cancer, may serve as a novel and effective therapeutic target. More importantly, our study clearly demonstrates a very promising therapeutic potential of cyclin E siRNA for treating the cyclin E-overexpressing breast cancers, including the very malignant triple-negative breast cancers

BRIT1/MCPH1 Is Essential for Mitotic and Meiotic Recombination DNA Repair and Maintaining Genomic Stability in Mice

BRIT1 protein (also known as MCPH1) contains 3 BRCT domains which are conserved in BRCA1, BRCA2, and other important molecules involved in DNA damage signaling, DNA repair, and tumor suppression. BRIT1 mutations or aberrant expression are found in primary microcephaly patients as well as in cancer patients. Recent in vitro studies suggest that BRIT1/MCPH1 functions as a novel key regulator in the DNA damage response pathways. To investigate its physiological role and dissect the underlying mechanisms, we generated BRIT1−/− mice and identified its essential roles in mitotic and meiotic recombination DNA repair and in maintaining genomic stability. Both BRIT1−/− mice and mouse embryonic fibroblasts (MEFs) were hypersensitive to γ-irradiation. BRIT1−/− MEFs and T lymphocytes exhibited severe chromatid breaks and reduced RAD51 foci formation after irradiation. Notably, BRIT1−/− mice were infertile and meiotic homologous recombination was impaired. BRIT1-deficient spermatocytes exhibited a failure of chromosomal synapsis, and meiosis was arrested at late zygotene of prophase I accompanied by apoptosis. In mutant spermatocytes, DNA double-strand breaks (DSBs) were formed, but localization of RAD51 or BRCA2 to meiotic chromosomes was severely impaired. In addition, we found that BRIT1 could bind to RAD51/BRCA2 complexes and that, in the absence of BRIT1, recruitment of RAD51 and BRCA2 to chromatin was reduced while their protein levels were not altered, indicating that BRIT1 is involved in mediating recruitment of RAD51/BRCA2 to the damage site. Collectively, our BRIT1-null mouse model demonstrates that BRIT1 is essential for maintaining genomic stability in vivo to protect the hosts from both programmed and irradiation-induced DNA damages, and its depletion causes a failure in both mitotic and meiotic recombination DNA repair via impairing RAD51/BRCA2's function and as a result leads to infertility and genomic instability in mice

SSTR5 P335L monoclonal antibody differentiates pancreatic neuroendocrine neuroplasms with different SSTR5 genotypes

Background: Somatostatin receptor type 5 (SSTR5) P335L is a hypofunctional, single nucleotide polymorphism of SSTR5 with implications in the diagnostics and therapy of pancreatic neuroendocrine neoplasms. The purpose of this study is to determine whether a SSTR5 P335L-specific monoclonal antibody could sufficiently differentiate pancreatic neuroendocrine neoplasms (PNENs) with different SSTR5 genotypes. Methods: Cellular proliferation rate, SSTR5 mRNA level, and SSTR5 protein level were measured by performing MTS assay, a quantitative reverse transcription polymerase chain reaction study, Western blot analysis, and immunohistochemistry, respectively. SSTR5 genotype was determined with the TaqMan SNP Genotyping assay (Applied Biosystems, Foster City, CA). Results: We found that the SSTR5 analogue RPL-1980 inhibited cellular proliferation of CAPAN-1 cells more than that of PANC-1 cells. Only PANC-1 (TT) cells, but not CAPAN-1 (CC) cells expressed SSTR5 P335L. In 29 white patients with PNENs, 38% had a TT genotype for SSTR5 P335L, 24% had a CC genotype for WT SSTR5, and 38% hada CT genotype for both SSTR5 P335L and WT SSTR5. Immunohistochemistry using SSTR5 P335L monoclonal antibody detected immunostaining signals only from the neuroendocrine specimens with TT and CT genotypes, but not those with CC genotypes. Conclusion: A SSTR5 P335L monoclonal antibody that specifically recognizes SSTR5 P335L but not WT SSTR5 could differentiate PNENs with different SSTR5 genotypes, thereby providing a potential tool for the clinical diagnosis of PNEN. © 2011 Published by Mosby, Inc

Molecular analysis of <i>in vivo</i> tumor samples.

<p><b>A.</b> Electropherogram analyze % of mu and wt <i>KRAS</i> transcripts in <i>in vivo</i> treated tumor samples. Tumors were removed from animals after four weeks of various treatments, proportions of mu and wt <i>KRAS</i> transcripts were analyzed by RFLP and assayed by Experion. Sample designation is the same as indicated for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193644#pone.0193644.g004" target="_blank">Fig 4B</a>. % mu and wt <i>KRAS</i> transcripts show at the bottom of the figure was determined by Experion software. <b>B.</b> Western transfer shows protein expression in various tumor samples. Numbers on each sample indicate treatment groups as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193644#pone.0193644.g004" target="_blank">Fig 4</a>. Two independent isolated tumors are analyzed for each treatment group. Panel a shows RAS protein in various treated tumor samples normalized against GAPDH. Panel b shows p-EGFR at position Y1068 quantitatively normalized against total EGFR protein. Panel c shows total EGFR protein for various treatment groups. <b>C.</b> Bar graphs summarize fold intensity difference from various groups of <i>in vivo</i> samples. Sample groupings are the same as shown on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193644#pone.0193644.g004" target="_blank">Fig 4</a>. Panel a is for p-EGFR at Y1045 normalized to total EGFR protein. Panel b is for p-EGFR at Y1068 normalized to total EGFR protein. Panel c is for p-EGFR at Y1125 normalized to total EGFR protein. Panel d is total EGFR protein normalized to GAPDH. For each sample n = 3. Bar graphs shown are data obtained from approximately half of tumor of three independent animals. Standard deviation bar represents measurements of tumor from three animals. With one tailed, equal variances, student T-test, the following samples show statistical significant ρ-value ≤ 0.05: Panel a between samples 1 and 6, Panel c between samples 1 and 5, Panel d between samples 1 and 4, 2 and 4, 2 and 6.</p

PANC-1 xenograft model.

<p><b>A.</b> Average tumor volume measurement of PANC-1 tumor xenograft. Group 1: no treatment (blue line), Group 2: vehicle treated (red line), Group 3: 5 μg of 131 (DVR triple knockdown in miR17-92 backbone, green line), Group 4: 25 μg of 131 (DVR triple knockdown in miR17-92 backbone, light blue line), Group 5: 5 μg of 132 (CDV triple knockdown in miR17-92 backbone, purple line), Group 6: 25 μg of 132 (CDV triple knockdown in miR17-92 backbone, dark red line). <b>B.</b> Bar graph show average copy number of plasmids per 100 ng of genomic DNA found in tumor samples. The same treatment grouping as for panel A, samples A, B, or C represents three different tumors from three different animals of the same treatment group.</p

Triple mutant knockdown constructs and mutant allele selective knockdown.

<p><b>A</b>. Schematics shown expression unit sequence arrangement of triple knockdown constructs in either miR30a backbones or miR-17-92 cluster backbones. <b>B.</b> Bar graphs show analysis of the specificity of triple knockdown constructs by reporter vectors. For each bar graph, the Y-axis is FF/RL RLI ratio (mu/wt) and the X-axis is knockdown vectors tested. At the top of each panel indicate individual mutant targeting vectors tested. Samples from left to right: C = control, G12D = G12D knockdown vector (three independent knockdown vectors with different efficiencies; G12D specific knockdown vector with mutated nucleotide at position 2, position 3 and position 6 of the guide strand, respectively), G12V = G12V knockdown vector with mutated nucleotide at position 4 of the guide strand, G12C = G12C knockdown vector with mutated nucleotide at position 3 of the guide strand, G12R = G12R knockdown vector with mutated nucleotide at position 4 of the guide strand, DVR = triple knockdown vectors for G12D, G12V and G12R in miR30a backbone (code named 129), CDV = triple knockdown vectors for G12C, G12D and G12V in miR30a backbone (code named 130), DVR = triple knockdown vectors for G12D, G12V and G12R in miR17-92 backbone (code named 131), CDV = triple knockdown vectors for G12C, G12D and G12V in miR17-92 backbone (code named 132). The mutated nucleotide position at the guide strand of the triple constructs for G12D, G12V, G12C and G12R were guide strand position 3, 4, 3 and 4, respectively. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post-transfection. <b>C.</b> Elecropherogram show RFLP of <i>KRAS</i> mRNA in PANC-1 cells stably transformed with triple knockdown vectors. Each individual lane is labeled with their respective % of mutant vs. wild <i>KRAS</i> transcripts. % of mutant vs. wild-type was assessed by electrophaerogram band intensity scan. Samples PANC1 = non-transformed parent PANC-1 cells, Empty vector = non-transformed parent PANC-1 cells transfected with empty vector (pUMVC3 with no insert), Triple DVR (129) = PANC-1 cells transformed with DVR triple knockdown vector in miR-30a backbone (code named 129), Triple CDV (130) = PANC-1 cells transformed with CDV triple knockdown vector in miR-30a backbone (code named 130), Triple DVR (131) = PANC-1 cells transformed with DVR triple knockdown vector in miR-17-92 backbone (code named 131), Triple CDV (132) = PANC-1 cells transformed with CDV triple knockdown vector in miR-17-92 backbone (code named 132), G12V = PANC-1 cells transformed with G12V knockdown vector.</p

<i>In vivo</i> treatment show no adverse effect with PANC-1 xenograft model.

<p>Average body weight of animal with the same grouping as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193644#pone.0193644.g004" target="_blank">Fig 4A</a>.</p

Dual luciferase system to identify the most optimum mutant selective constructs.

<p>Coding sequences for the first 17 amino acids of <i>KRAS</i> wild-type (wt) and mutant (mu) were inserted into the amino terminus of the psiCHECK2’s hRluc (renilla) and hluc (firefly) coding sequence, respectively. Knockdown of wt vs. mu sequence is compared by renilla to firefly intensity ratio. <b>A.</b> Schematic of the sequence insertion into psiCHECK2 for reporter constructs. <b>B.</b> Bar graph show comparison of reporter constructs relative light unit (RLU) intensity ratio of renilla (RL) to firefly (FF). Y-axis is RL/FF RLU ratio. X-axis is reporter test vectors and parent reporter vector. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. <b>C.</b> Positional effect of G12D knockdown constructs; panel <b>a</b>: Table illustrate each constructs guide strand sequence in relation to G12D mutation site. 1^st column indicates position of G12D mutation in guide strand of each construct. 2^nd column is the code for each construct. The guide strand sequence is shown as the complement of target sequence at 3’ to 5’ orientation; panel <b>b</b>: Bar graph show comparative plot of FF/RL RLU ratio (mu/wt) for each knockdown construct. Sample C is the control without knockdown vector. The red bar represents average control sample value for visual enhancement. Standard deviation bar represents measurement from quadruplet samples of independently transfected cells in 96-well format and assayed simultaneously post transfection. Two-tailed student T-test indicates ρ-value ≤ 0.05 between control and samples 86, 87, 88, 75, 76 and 77. <b>D.</b> G12D, G12V, G12R and G12C knockdown constructs (with mutation nucleotide at position 3 or 4 of the guide strand) were tested against test reporter vectors of all four mutations. Bar graph shows the summary of relative average FF/RL RLU ratio (mu/wt). * indicate the most effective constructs for each mutation. X-axis is the knockdown constructs for G12D, G12V, G12R and G12C. P3 indicate knockdown construct with mutated nucleotide at position 3 of the guide strand. P4 indicate knockdown construct with mutated nucleotide at position 4 of the guide strand. Y-axis is the FF/RL RLU ratio.</p

Physical properties of reconstituted material comparison.

<p>Physical properties of reconstituted material comparison.</p