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1.  Recurrent read-through fusion transcripts in breast cancer 
Read-through fusion transcripts that result from the splicing of two adjacent genes in the same coding orientation are a recently discovered type of chimeric RNA. We sought to determine if read-through fusion transcripts exist in breast cancer. We performed paired-end RNA-seq of 168 breast samples, including 28 breast cancer cell lines, 42 triple negative breast cancer primary tumors, 42 estrogen receptor positive (ER+) breast cancer primary tumors, and 56 non-malignant breast tissue samples. We analyzed the sequencing data to identify breast cancer associated read-through fusion transcripts. We discovered two recurrent read-through fusion transcripts that were identified in breast cancer cell lines, confirmed across breast cancer primary tumors, and were not detected in normal tissues (SCNN1A-TNFRSF1A and CTSD-IFITM10). Both fusion transcripts use canonical splice sites to join the last splice donor of the 5′ gene to the first splice acceptor of the 3′ gene, creating an in-frame fusion transcript. Western blots indicated that the fusion transcripts are translated into fusion proteins in breast cancer cells. Custom small interfering RNAs targeting the CTSD-IFITM10 fusion junction reduced expression of the fusion transcript and reduced breast cancer cell proliferation. Read-through fusion transcripts between adjacent genes with different biochemical functions represent a new type of recurrent molecular defect in breast cancer that warrant further investigation as potential biomarkers and therapeutic targets. Both breast cancer associated fusion transcripts identified in this study involve membrane proteins (SCNN1A-TNFRSF1A and CTSD-IFITM10), which raises the possibility that they could be breast cancer-specific cell surface markers.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-014-3019-2) contains supplementary material, which is available to authorized users.
doi:10.1007/s10549-014-3019-2
PMCID: PMC4085473  PMID: 24929677
2.  HER2 overexpression renders human breast cancers sensitive to PARP inhibition independently of any defect in homologous recombination DNA repair 
Cancer research  2012;72(18):4796-4806.
HER2 overexpression in breast cancer confers increased tumor aggressiveness. Although anti-HER2 therapies against have improved patient outcome, resistance ultimately occurs. Poly(ADP-Ribose) polymerase (PARP) inhibitors target homologous recombination (HR)-deficient tumors, such as the BRCA-associated breast and ovarian cancers. In this study, we show that HER2+ breast cancers are susceptible to PARP inhibition independent of a HR deficiency. HER2 overexpression in HER2 negative breast cancer cells was sufficient to render cells susceptible to the PARP inhibitors ABT-888 and AZD-2281 both in vitro and in vivo, which was abrogated by HER2 reduction. In addition, ABT-888 significantly inhibited NFκB (p65/RelA) transcriptional activity in HER2+ but not HER2 negative breast cancer cells. This corresponded with a reduction in phosphorylated p65 and total IKKα levels, with a concomitant increase in IκBα. Overexpression of p65 abrogated cellular sensitivity to ABT-888, while IκBα overexpression reduced cell viability to a similar extent as ABT-888. Therefore, susceptibility of HER2+ breast cancer cells to PARP inhibition may be due to inhibition of NF-kB signaling driven by HER2. Our findings indicate that PARP inhibitors may be a novel therapeutic strategy for sporadic HER2 positive breast cancer patients.
doi:10.1158/0008-5472.CAN-12-1287
PMCID: PMC3458582  PMID: 22987487
breast cancer; HER2; PARP inhibitors; NFκB; homologous recombination repair
3.  Effect of anti-DR5 and chemotherapy on basal-like breast cancer 
The purpose is to evaluate sensitivity of basal-like breast cancer to treatment with anti-DR5 alone and in combination with chemotherapy. Cytotoxicity of TRA-8 anti-DR5 alone and in combination with doxorubicin or paclitaxel was examined. The role of a DR5-associated molecule (DDX3) in the regulation of apoptosis by recruitment of cIAP1 to the DR5/DDX3 complex was studied. SUM159 and 2LMP orthotopic xenografts were treated with TRA-8 alone and in combination with Abraxane or doxorubicin, and tumor growth inhibition determined. Diffusion-weighted magnetic resonance imaging was used to monitor early tumor response. The majority (12/15) of basal-like cell lines were very sensitive to TRA-8-induced cytotoxicity (IC50 values of 1.0–49 ng/ml). In contrast, 8/11 luminal or HER2-positive cell lines were resistant (IC50 > 1,000 ng/ml). Enhanced killing of basal-like cell lines was produced by combination treatment with TRA-8 and doxorubicin. Majority of basal cell lines expressed lower levels of DR5-associated DDX3 and cIAP1 than luminal and HER2-positive cell lines. TRA-8 inhibited growth of basal xenografts and produced 20% complete 2LMP tumor regressions. TRA-8 and chemotherapy produced greater 2LMP growth inhibition than either alone. An increase in apparent diffusion coefficient in 2LMP tumors was measured in a week of therapy with TRA-8 and Abraxane. Basal-like cell lines were more sensitive to TRA-8-mediated cytotoxicity than HER2-over-expressing and luminal cell lines, and chemotherapy enhanced cytotoxicity. High sensitivity of basal cells to TRA-8 correlated with low expression of DR5/DDX3/cIAP1 complex. Treatment with TRA-8 and chemotherapy may be an effective therapy for basal-like breast cancer.
doi:10.1007/s10549-011-1755-0
PMCID: PMC3613128  PMID: 21901385
Basal-like breast cancer; Anti-DR5 antibody; Chemotherapy
4.  Basal-like breast cancer stem cells are sensitive to anti-DR5 mediated cytotoxicity 
Breast cancer stem cells (BrCSC) are resistant to common therapeutic modalities including chemotherapy, radiation, and hormonal agents. They are thought to contribute to treatment resistance, relapse, and metastases. This study examines the effect of a monoclonal anti-DR5 antibody (TRA-8) and chemotherapy (adriamycin, taxol) on BrCSC populations from basal-like breast cancer cell lines. Doubly enriched BrCSC (CD44+, CD24−, ALDH+) cells were exposed to TRA-8 and control reagents and examined for cytotoxicity, caspase activation, tumorsphere formation and tumorigenicity. Doubly enriched BrCSC populations expressed cell surface DR5 and were sensitive to TRA-8 mediated cytotoxicity with induction of caspase 8 and 3 activation. TRA-8 at sub-nanomolar concentrations inhibited 2LMP and SUM159 BrCSC tumorsphere formation and was more than 50-fold more inhibitory than TRAIL or anti-DR4 at equimolar concentrations. Chemotherapy treatment of 2LMP and SUM159 cell lines resulted in a relative increase of BrCSC, whereas TRA-8 produced a decrease in the percentage of BrCSC. TRA-8 exposure to 2LMP and SUM159 BrCSC preparations produced significant inhibition of tumorigenicity. DR5 maybe a therapeutic target on the surface of basal-like BrCSC which is amenable to agonistic monoclonal anti-DR5 therapy.
doi:10.1007/s10549-011-1763-0
PMCID: PMC3609658  PMID: 21915634
Anti-DR5; Tigatuzumab; Basal-like breast cancer; Breast cancer stem cells; Tumor initiating cells; Tumorspheres; Death receptor 5
5.  Cellular Model of Warburg Effect Identifies Tumor Promoting Function of UCP2 in Breast Cancer and Its Suppression by Genipin 
PLoS ONE  2011;6(9):e24792.
The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho0) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho0 derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho0 breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.
doi:10.1371/journal.pone.0024792
PMCID: PMC3174207  PMID: 21935467
6.  Cetuximab Augments Cytotoxicity with Poly (ADP-Ribose) Polymerase Inhibition in Head and Neck Cancer 
PLoS ONE  2011;6(8):e24148.
Overexpression of the epidermal growth factor receptor (EGFR) is a hallmark of head and neck cancers and confers increased resistance and inferior survival rates. Despite targeted agents against EGFR, such as cetuximab (C225), almost half of treated patients fail this therapy, necessitating novel therapeutic strategies. Poly (ADP-Ribose) polymerase (PARP) inhibitors (PARPi) have gained recent attention due to their unique selectivity in killing tumors with defective DNA repair. In this study, we demonstrate that C225 enhances cytotoxicity with the PARPi ABT-888 in UM-SCC1, UM-SCC6, and FaDu head and neck cancer cells. The mechanism of increased susceptibility to C225 and PARPi involves C225-mediated reduction of non-homologous end-joining (NHEJ)- and homologous recombination (HR)-mediated DNA double strand break (DSB) repair, the subsequent persistence of DNA damage, and activation of the intrinsic apoptotic pathway. By generating a DSB repair deficiency, C225 can render head and neck tumor cells susceptible to PARP inhibition. The combination of C225 and the PARPi ABT-888 can thus be an innovative treatment strategy to potentially improve outcomes in head and neck cancer patients. Furthermore, this strategy may also be feasible for other EGFR overexpressing tumors, including lung and brain cancers.
doi:10.1371/journal.pone.0024148
PMCID: PMC3166164  PMID: 21912620
7.  Epithelial transformation by KLF4 requires Notch1 but not canonical Notch1 signaling 
Cancer biology & therapy  2009;8(19):1840-1851.
The transcription factors Notch1 and KLF4 specify epithelial cell fates and confer stem cell properties. suggesting a functional relationship, each gene can act to promote or suppress tumorigenesis in a context-dependent manner, and alteration of KLF4 or Notch pathway genes in mice gives rise to similar phenotypes. Activation of a conditional allele of KLF4 in RK3E epithelial cells rapidly induces expression of Notch1 mRNA and the active, intracellular form of Notch1. KLF4-induced transformation was suppressed by knockdown of endogenous Notch1 using siRNA or an inhibitor of γ-secretase. Chromatin immunoprecipitation assay shows that KLF4 binds to the proximal Notch1 promoter in human mammary epithelial cells, and siRNA-mediated suppression of KLF4 in human mammary cancer cells results in reduced expression of Notch1. Furthermore, KLF4 and Notch1 expression are correlated in primary human breast tumors (N = 89; pearson analysis, r > 0.5, p < 0.0001). Like KLF4, Notch1 was previously shown to induce transformation of rat cells immortalized with adenovirus E1A, similar to RK3E cells. We therefore compared the signaling requirements for Notch1- or KLF4-induced malignant transformation of RK3E. As expected, transformation by Notch1 was suppressed by dominant-negative CSL or MaML1, inhibitors of canonical Notch1 signaling. However, these inhibitors did not suppress transformation by KLF4. Therefore, while KLF4-induced transformation requires Notch1, canonical Notch1 signaling is not required, and Notch1 may signal through a distinct pathway in cells with increased KLF4 activity. These results suggest that KLF4 could contribute to breast tumor progression by activating synthesis of Notch1 and by promoting signaling through a non-canonical Notch1 pathway.
PMCID: PMC2795010  PMID: 19717984
KLF4; Notch1; transcription factor; breast cancer; malignant transformation; epithelial cell; carcinoma
8.  Treatment of Human Colon Cancer Xenografts with TRA-8 Anti-death Receptor 5 Antibody Alone or in Combination with CPT-11 
Purpose
This study was designed to evaluate the in vitro cytotoxicity and in vivo efficacy of TRA-8, a mouse monoclonal antibody that binds to the DR5 death receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L), alone and in combination with CPT-11 against human colon cancer cells and xenografts.
Experimental Design
DR5 expression was assessed on human colon cancer cell lines using flow cytometry, and cellular cytotoxicity following TRA-8 treatment, alone and in combination with SN-38 was determined by measuring cellular ATP levels. Tumor growth inhibition and regression rates of well-established s.c. COLO 205, SW948, HCT116 and HT-29 colon cancer xenografts in athymic nude mice treated with TRA-8 or CPT-11 alone and in combination were determined. 99mTc-TRA-8 was utilized to examine tumor localization of TRA-8 in animals bearing each of the 4 xenografts. In addition, whole body biodistribution and imaging was carried out in COLO 205 bearing animals using in vivo SPECT imaging and tissue counting.
Results
DR5 expression was highest on HCT116, intermediate on SW948 and COLO 205 cells, and lowest on HT-29 cells. COLO 205 cells were the most sensitive to TRA-8-induced cytotoxicity in vitro, SW948 and HCT116 cell lines were moderately sensitive, and HT-29 cells were resistant. Combination treatment with TRA-8 and SN-38 produced additive to synergistic cytotoxicity against all cell lines compared to either single agent. The levels of apoptosis in all cell lines, including HT-29, were increased by combination treatment with SN-38. In vivo, combination therapy with TRA-8 and CPT-11 was superior to either single agent regimen for three of the xenografts; COLO 205, SW948, and HCT116. COLO 205 tumors were most responsive to therapy with 73% complete regressions following combination therapy. HT-29 cells derived no anti-tumor efficacy from TRA-8 therapy. Tumor xenografts established from the 4 colon cancer cell lines had comparable specific localization of 99mTc-TRA-8.
Conclusions
In vitro and in vivo effects of TRA-8 anti-DR5 monoclonal antibody on four different colon cancer cell lines and xenografts were quite variable. The HT-29 cell line had low surface DR5 expression and was resistant to TRA-8 both in vitro and in vivo. Three cell lines (COLO 205, SW948, and HCT116) exhibited moderate to high sensitivity to TRA-8 mediated cytotoxicity which was further enhanced by the addition of SN-38, the active metabolite of CPT-11. In vivo, the combination of TRA-8 and CPT-11 treatment produced the highest anti-tumor efficacy against xenografts established from the three TRA-8 sensitive tumor cell lines. All 4 colon cancer xenografts had comparable localization of 99mTc-TRA-8. These studies support the strategy of TRA-8/CPT-11 combined treatment in human colon cancer clinical trials.
doi:10.1158/1078-0432.CCR-07-1392
PMCID: PMC2676875  PMID: 18381960
9.  Human Monocyte Antibody-Dependent Cell-Mediated Cytotoxicity to Tumor Cells 
Journal of Clinical Investigation  1978;62(6):1172-1180.
Previous investigations of mononuclear cell antibody-dependent cell-mediated cytotoxicity (ADCC) toward tumor cells suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. This report, however, demonstrates that human monocytes are able to carry out ADCC toward three different human tumor cell lines (CEM T lymphoblasts, Raji bone marrow-derived (B) lymphoblasts, and HeLa cells). The cytolytic event was found to be temperature dependent and rapid, with most of the lysis occurring in the first 4 h of incubation. The extent of lysis was directly related to the number of monocytes (effector cells) and to the degree of antibody sensitization of the target cells. The antibody-dependent cell contact-mediated nature of the cytolytic event was confirmed by inhibition with competing nonspecific monomeric immunoglobulin and by the ability of monocytes in “innocent bystander” experiments to lyse antibody-coated targets but not nonantibody-coated target cells. Evidence that monocytes were clearly the effector cells in the monocyte preparations included the observation that preincubation of effector cells with opsonized zymosan particles abolished ADCC by monocytes, but had little effect on lymphocyte ADCC. Furthermore, no evidence for Fc receptor K lymphocyte contamination of the monocyte preparations was found using antibody-coated target cells that were selectively lysed by lymphocytes but not monocytes. We suggest that ADCC toward tumor cell targets may prove to be a useful assay of monocyte function in normal and disease states.
PMCID: PMC371881  PMID: 748372
10.  Effects of Corticosteroids on Human Monocyte Function 
Journal of Clinical Investigation  1974;54(6):1337-1343.
This report examined the effect of corticosteroids in vitro on human peripheral blood monocytes, essential cells in both immune and nonimmune cellular defense mechanisms. Monocyte chemotaxis in response to sera, Escherichia coli filtrate, and lymphokine chemotactic factor was markedly reduced (P < 0.01) by hydrocortisone succinate (HCS) at 16 μg/ml. Methylprednisolone succinate and unesterified hydrocortisone produced similar impairment of monocyte chemotaxis while two drugs which unmodified do not enter cells, hydrocortisone phosphate (HCP) and cortisone acetate, had no effect on chemotaxis. HCS also significantly impaired monocyte random migration at 16 μg/ml. Monocyte bactericidal activity was reduced by HCS at 16 μg/ml (P < 0.01)) but was not affected by HCP even at 120 μg/ml. In comparison, HCS did not alter granulocyte chemotaxis even at 500 μg/ml, and bactericidal activity was reduced at 16 μg/ml (P < 0.01). Monocyte phagocytosis of cryptococci was reduced only 20% (P < 0.05) at 120 μg/ml. HCS at 120 μg/ml did not alter monocyte base-line or postphagocytic hexosemonophosphate shunt activity, viability by trypan blue exclusion, adherence to tissue culture flasks, or surface binding of IgG globulin. These corticosteroid-induced defects in monocyte function may contribute to reduced cellular defense during corticosteroid therapy.
PMCID: PMC301688  PMID: 4612058
11.  Immunologic Specificity of Transfer Factor 
Journal of Clinical Investigation  1974;54(4):997-1000.
This study examined the immunologic specificity of transfer factor using a chromatographically purified transfer factor preparation. The specificity of transfer was examined utilizing immunity to keyhole limpet hemocyanin (KLH) and tuberculin. Transfer factor prepared from a donor immune to KLH successfully transferred KLH skin test reactivity to 10 out of 10 recipients. In contrast, comparable amounts of transfer factor from two donors not immune to KLH failed to transfer immunity to KLH in 11 recipients despite evidence for successful transfer of tuberculin reactivity. Unlike prior studies with a variety of antigens, the immunity to KLH in recipients of KLH immune transfer factor appeared comparable to that of the donor since both could be elicited with the same skin test antigen dose. These observations indicate that transfer factor can initiate a specific immune response to an antigen not previously encountered by the recipient and that in certain circumstances this immune response can be comparable to that of the donor. These observations on specificity and potency of transfer factor have important implications for the clinical use of this material.
PMCID: PMC301641  PMID: 4139171

Results 1-11 (11)