Search

PDF file - 73K

PDF file - 81K

PDF file - 221K, E-cadherin and beta-catenin expression in SKBR3 and SKBR3/100-8 cells

PDF file - 53K, Confirmation of Wnt pathway gene expression by RT-Q-PCR

PDF file - 1MB, Pathway signaling differentially expressed in SKBR3/100-8 compared to SKBR3 revealed by IPA

PDF file - 43K, siRNA knocked down EGFR in SKBR3/100-8 cells

PDF file - 59K, siRNA knocked down HER3 sensitizes SKBR3/100-8 cell response to trastuzumab-induced cell growth inhibition

Supplementary Figure 4 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Supplementary Figure 1 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Supplementary Table 1 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide–induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356–65]

Supplementary Figure 3 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Supplementary Methods, Figure Legends 1-4 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

Supplementary Figure 2 from The Cell Death–Inducing Activity of the Peptide Containing Noxa Mitochondrial-Targeting Domain Is Associated with Calcium Release

The abnormal PI3K/AKT/mTOR pathway is one of the most common genomic abnormalities in breast cancers including triple-negative breast cancer (TNBC), and pharmacologic inhibition of these aberrations has shown activity in TNBC patients. Here, we designed and identified a small-molecule Comp34 that suppresses both AKT and mTOR protein expression and exhibits robust cytotoxicity towards TNBC cells but not nontumorigenic normal breast epithelial cells. Mechanically, long noncoding RNA (lncRNA) AL354740.1-204 (also named as NUDT3-AS4) acts as a microRNA sponge to compete with AKT1/mTOR mRNAs for binding to miR-99s, leading to decrease in degradation of AKT1/mTOR mRNAs and subsequent increase in AKT1/mTOR protein expression. Inhibition of lncRNA-NUDT3-AS4 and suppression of the NUDT3-AS4/miR-99s association contribute to Comp34-affected biologic pathways. In addition, Comp34 alone is effective in cells with secondary resistance to rapamycin, the best-known inhibitor of mTOR, and displays a greater in vivo antitumor efficacy and lower toxicity than rapamycin in TNBC xenografted models. In conclusion, NUDT3-AS4 may play a proproliferative role in TNBC and be considered a relevant therapeutic target, and Comp34 presents promising activity as a single agent to inhibit TNBC through regulation of NUDT3-AS4 and miR-99s.

Scope We investigated whether a combination of two promising chemopreventive agents arctigenin (Arc) and quercetin (Q) increases the anticarcinogenic potency at lower concentrations than necessary when used individually in prostate cancer. Methods and results Androgen-dependent LAPC-4 and LNCaP prostate cancer cells were treated with low doses of Arc and Q alone or in combination for 48 h. The antiproliferative activity of Arc was 10- to 20-fold stronger than Q in both cell lines. Their combination synergistically enhanced the antiproliferative effect, with a stronger effect in androgen receptor (AR) wild-type LAPC-4 cells than in AR mutated LNCaP cells. Arc demonstrated a strong ability to inhibit AR protein expression in LAPC-4 cells. The combination treatment significantly inhibited both AR and PI3K/Akt pathways compared to control. A protein array analysis revealed that the mixture targets multiple pathways particularly in LAPC-4 cells including Stat3 pathway. The mixture significantly inhibited the expression of several oncogenic microRNAs including miR-21, miR-19b, and miR-148a compared to control. The mixture also enhanced the inhibition of cell migration in both cell lines compared to individual compounds tested. Conclusion The combination of Arc and Q that target similar pathways, at low physiological doses, provides a novel regimen with enhanced chemoprevention in prostate cancer.

To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors, we created trastuzumab-insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression and show increase in EGF receptor (EGFR). Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab-resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab-resistant cells also promoted a partial EMT-like transition (epithelial-to-mesenchymal transition); increased N-cadherin, Twist, Slug; and decreased E-cadherin. Knockdown of Wnt3 by siRNA restored cytoplasmic expression of β-catenin and decreased EGFR expression in trastuzumab-resistant cells. Furthermore, the EMT markers were decreased, E-cadherin was increased, and the cell invasiveness was inhibited in response to the Wnt3 downregulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed a significant decrease in E-cadherin and increase in N-cadherin, Twist, and Slug. The cells were less sensitive to trastuzumab than parental SKBR3 and vector-transfected cells. In summary, our data suggest that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2-overexpressing breast cancer cells. Mol Cancer Res; 10(12); 1597–606. ©2012 AACR.

DNA damage stabilizes the p53 tumor suppressor protein that determines the cell fate by either cell cycle arrest or cell death induction. Noxa, the BH3-only Bcl-2 family protein, was shown to be a key player in p53-induced cell death through the mitochondrial dysfunction; however, the molecular mechanism by which Noxa induces the mitochondrial dysfunction to cause cell death in response to genotoxic agents is largely unknown. Here, we show that the mitochondrial-targeting domain (MTD) of Noxa is a prodeath domain. Peptide containing MTD causes massive necrosis in vitro through cytosolic calcium increase; it is released from the mitochondria by opening the mitochondrial permeability transition pore. MTD peptide–induced cell death can be inhibited by calcium chelator BAPTA-AM. Moreover, MTD peptide shows the potent tumor-killing activities in mice by joining with tumor-homing motifs. [Cancer Res 2009;69(21):8356–65]

Colorectal cancer (CRC) is one of the three leading causes for cancer mortality. CRC kills over 600,000 people annually worldwide. The most common cause of death from CRC is the metastasis to distant organs. However, biomarkers for CRC metastasis remain ill-defined. We compared primary and metastatic CRC cell lines for their angiogenesis-protein profiles and intracellular signaling profiles to identify novel biomarkers for CRC metastasis. To this end, we used primary and metastatic CRC cell lines as a model system and normal human colon cell line as a control. The angiogenesis profiles two isogenic CRC cell lines, SW480 and SW620, and HT-29 and T84 revealed that VEGF was upregulated in both SW620 and T84 whereas coagulation factor III, IGFBP-3, DPP IV, PDGF AA/AB, endothelin I and CXCL16 were downregulated specifically in metastatic cell lines. Furthermore, we found that TIMP-1, amphiregulin, endostatin, angiogenin were upregulated in SW620 whereas downregulated in T84. Angiogenin was downregulated in T84 and GM-CSF was also downregulated in SW620. To induce CRC cell metastasis, we treated cells with pro-inflammatory cytokine IL-6. Upon IL-6 treatment, epithelial-mesenchymal transition was induced in CRC cells. When DLD-1 and HT-29 cells were treated with IL-6; Akt, STAT3, AMPKα and Bad phosphorylation levels were increased. Interestingly, SW620 showed the same signal activation pattern with IL-6 treatment of HT-29 and DLD-1. Our data suggest that Akt, STAT3, AMPKα and Bad activation can be biomarkers for metastatic colorectal cancer. IL-6 treatment specifically reduced phosphorylation levels of EGFR, HER2 receptor, Insulin R and IGF-1R in receptor tyrosine kinase array study with HT-29. Taken together, we have identified novel biomarkers for metastatic CRC through the angiogenesis-antibody array and intracellular signaling array studies. Present study suggests that those novel biomarkers can be used as CRC prognosis biomarkers, and as potential targets for the metastatic CRC therapy.

The Saccharomyces cerevisiae [PSI(+)] prion is believed to be a self-propagating cytoplasmic amyloid. Earlier characterization of HSP70 (SSA1) mutations suggested that [PSI(+)] propagation is impaired by alterations that enhance Ssa1p's substrate binding. This impairment is overcome by second-site mutations in Ssa1p's conserved C-terminal motif (GPTVEEVD), which mediates interactions with tetratricopeptide repeat (TPR) cochaperones. Sti1p, a TPR cochaperone homolog of mammalian Hop1 (Hsp70/90 organizing protein), activates Ssa1p ATPase, which promotes substrate binding by Ssa1p. Here we find that in SSA1-21 cells depletion of Sti1p improved [PSI(+)] propagation, while excess Sti1p weakened it. In contrast, depletion of Fes1p, a nucleotide exchange factor for Ssa1p that facilitates substrate release, weakened [PSI(+)] propagation, while overproducing Fes1p improved it. Therefore, alterations of Hsp70 cochaperones that promote or prolong Hsp70 substrate binding impair [PSI(+)] propagation. We also find that the GPTVEEVD motif is important for physical interaction with Hsp40 (Ydj1p), another Hsp70 cochaperone that promotes substrate binding but is dispensable for viability. We further find that depleting Cpr7p, an Hsp90 TPR cochaperone and CyP-40 cyclophilin homolog, improved [PSI(+)] propagation in SSA1 mutants. Although Cpr7p and Sti1p are Hsp90 cochaperones, we provide evidence that Hsp90 is not involved in [PSI(+)] propagation, suggesting that Sti1p and Cpr7p functionally interact with Hsp70 independently of Hsp90.

We studied a Cryptococcus neoformans strain that caused feline chronic nasal granuloma without disseminated disease. This strain, B-4551, grows at temperatures up to 35°C and fails to cause systemic infection in mice. Many cells of B-4551 formed short hyphal elements in feline nasal tissue and occasionally at 35°C in vitro. A complementation and sequence analysis revealed that the temperature-sensitive (Ts) phenotype of B-4551 was due to deletion of a lysine residue in the cryptococcal CCN1 gene. B-4551 complemented with the wild type CCN1 gene grew at 37°C and caused fatal systemic infection in mice. The CCN1 gene encodes a protein containing 16 copies of a tetratricopeptide repeat. CCN1 is homologous to the Saccharomyces cerevisiae CLF1 gene, which is required for pre-mRNA splicing, cell cycle progression, and DNA replication, and to the Drosophila melanogaster crn gene, which is involved in neurogenesis. CLF1 complemented the Ts phenotype of B-4551. CCN1 , however, failed to rescue the clf1 mutant in S. cerevisiae . These results indicate that the Ccn1p may not be as functionally diverse as Clf1p in yeast.

The Drosophila crooked neck (crn) gene is essential for embryogenesis and has been implicated in cell cycle progression and in pre-mRNA splicing although a direct role in either process has not been established. Here we report isolation of the human crooked neck homolog, HCRN, and provide evidence for its function in splicing. HCRN encodes an unusual protein composed largely of tetratricopeptide repeat (TPR) elements. The crooked neck protein co-localizes with the SR and Sm protein splicing factors in discrete subnuclear domains implicated in snRNP biogenesis. In vitro assembly experiments show that an 83 kDa hcrn isoform is stably recruited to splicing complexes coincident with the addition of the U4/U6.U5 tri-snRNP particle. Crooked neck activity appears essential as extracts depleted of hcrn fail to splice pre-mRNA. These and related data support the view that crooked neck is a phylogenetically conserved pre-mRNA splicing factor.

There is an increasing evidence of pro-inflammatory cytokines involvement in cancer development. Here, we found that two cytokines, IL-6 and TNF-α, induced colorectal cancer cells more active and invasive. Combined treatments of IL-6 and TNF-α phosphorylated transcription factors STAT3 in a synergistic manner. STAT3 and NF-κB physically interacted upon cytokine stimulation. Similarly, STAT1 hetero-dimerized with STAT3 and the binding affinity was enhanced with cytokine treatments. STAT3 bound the promoter region of human telomerase reverse transcriptase (hTERT), and IL-6 and TNF-α stimulation further enhanced this STAT3 binding affinity. Withaferin A, an anti-inflammatory steroidal lactone, inhibited the IL-6 and TNF-α induced cancer cell invasiveness and decreased colonosphere formation. Notably, withaferin A inhibited STAT3 phosphorylation and abolished the STAT3 and NF-κB interactions. STAT3 binding to hTERT promoter was inhibited and telomerase activity was decreased with the withaferin A treatments. Taken together, pro-inflammatory cytokines induced-cancer cell invasiveness is mediated by STAT3 regulated mechanism in colorectal cancer cells. Our study suggests the novel natural compound therapy for the metastatic colorectal cancer in clinical settings. Citation Format: Seyung Chung, Quincy Okobi, Debbie Adekoya, Jaydutt Vadgama. Targeting STAT3 and telomerase for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 111. doi:10.1158/1538-7445.AM2017-111

Mutants in the Drosophila crooked neck (crn) gene show an embryonic lethal phenotype with severe developmental defects. The unusual crn protein consists of sixteen tandem repeats of the 34 amino acid tetratricopeptide (TPR) protein recognition domain. Crn-like TPR elements are found in several RNA processing proteins, although it is unknown how the TPR repeats or the crn protein contribute to Drosophila development. We have isolated a Saccharomyces cerevisiae gene, CLF1, that encodes a crooked neck-like factor. CLF1 is an essential gene but the lethal phenotype of a clf1::HIS3 chromosomal null mutant can be rescued by plasmid-based expression of CLF1 or the Drosophila crn open reading frame. Clf1p is required in vivo and in vitro for pre-mRNA 5' splice site cleavage. Extracts depleted of Clf1p arrest spliceosome assembly after U2 snRNP addition but prior to productive U4/U6.U5 association. Yeast two-hybrid analyses and in vitro binding studies show that Clf1p interacts specifically and differentially with the U1 snRNP-Prp40p protein and the yeast U2AF65 homolog, Mud2p. Intriguingly, Prp40p and Mud2p also bind the phylogenetically conserved branchpoint binding protein (BBP/SF1). Our results indicate that Clf1p acts as a scaffolding protein in spliceosome assembly and suggest that Clf1p may support the cross-intron bridge during the prespliceosome-to-spliceosome transition.

The association of human papillomavirus (HPV) with cervical cancer, head and neck cancer is well established, but the involvement of HPV E6/E7 oncoproteins in breast cancer is more contentious. Previous studies suggest that high risk HPV E6/E7 oncoproteins inactivate two tumor suppressor proteins p53 and pRb signaling and increase cell proliferation. There is cumulative evidence that high risk HPV 16 E6/E7 oncoproteins may have causal roles in some human breast cancers. Moreover, some studies have identified HPV DNA in breast tissue and breast cancer specimens. Nevertheless, the route of transmission of HPV in breast cells has not been determined, and the mechanisms by which HPV involvement in breast cancer are not known. We used a non-tumorigenic (MCF12A) epithelial breast cell line transfected with HPV 16 to elucidate the role of oncoproteins E6/E7 in pro-proliferative and anti-apoptotic signaling pathways. We also used Tamoxifen treatment, which is an antagonist of the estrogen receptor in breast tissues through its active metabolite, 4hydroxytamoxifen. We demonstrate that HPV E6/E7 transfection activates the extracellular signal-regulated kinase (ERK) MAPK pathway and Akt/PKB kinase signaling pathways through activating estrogen receptor alpha in MCF12A cells. E6/E7 oncoproteins transfection alone dramatically increases estrogen receptor alpha protein expression. Treatment with tamoxifen, an antagonist of the ER, significantly abolishes E6/E7-increase in ERK and PKB phosphorylation without altering their protein levels, suggesting that the estrogen receptor is a key mediator of HPV E6/E7 mediated ERK and Akt/PKB activation. Our current work show an insightful understanding of the molecular mechanisms of HPV-elicited Akt/PKB activation and its roles in cell proliferation and survival in normal breast epithelial cells. Therefore, these findings indicate that HPV E6/E7 oncoproteins may promote the transforming activities of high-risk HPV E6/E7 proteins and cell proliferation in breast cells. Therefore, suppression of estrogen receptor signaling networks may be used as a therapeutic strategy for HPV associated lesions and cancers such as breast metastasis. Citation Format: Eva McGhee, Lucy Tran, Mengtao Li, Yong Wu, Seyung Chung, Cheryl Araniego, Karen Tate, Melanie Baker, Kamilah Evans, Mechelle Rouse, Jay Vadgama. Estrogen receptor alpha mediates HPV E6/E7 oncoproteins: Induced breast cells proliferation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4588.

Cyclophosphamide induced cystitis is an established model for the study of bladder injury and wound healing. Glycosylation is an important modification mechanism that regulates the structure and function of secreted proteins and growth factors from inflammation sites. We determined the effect of cyclophosphamide induced cystitis on O-GlcNAc mediated glycosylation in the bladder.Cystitis in WT C57BL6 mice was induced with intraperitoneal cyclophosphamide. Retrieved bladders were analyzed using histology, immunohistochemistry, reverse transcriptase-polymerase chain reaction and Western blot for glycosylation associated factors.Acute bladder injury was seen up to 168 hours (7 days) after injection. Reverse transcriptase-polymerase chain reaction revealed down-regulation of O-GlcNAc transferase, a key enzyme in O-GlcNAc mediated glycosylation, at the 8, 48 and 168-hour time points. Also, the glycosidase menangioma expressed antigen 5 was up-regulated at similar time points. Western blot analysis revealed decreased glycosylated protein during cyclophosphamide induced inflammation.To our knowledge we report the first study of alterations in O-GlcNAc mediated glycosylation activity in bladders with cyclophosphamide induced cystitis. Glycosylation may have a significant role in the bladder wound healing process. Future studies of the glycosylation signaling pathways in the bladder would assist in future potential therapy for bladder inflammatory disease and cancer by elucidating pathways that guide bladder development and wound healing.

The putative Cryptococcus neoformans pheromone receptor gene CPRα was isolated and studied for its role in mating and filamentation. CPRα is MATα specific and located adjacent to STE12α at the MATα locus. It encodes a protein which possesses high sequence similarity to the seven-transmembrane class of G-protein-coupled pheromone receptors reported for other basidiomycetous fungi. Strains containing a deletion of the CPRα gene exhibited drastic reductions in mating efficiency but were not completely sterile. Δcpr α cells displayed wild-type mating efficiency when reconstituted with the wild-type CPRα gene. Hyphal production on filament agar was not affected in the Δcprα strain, indicating no significant role for CPRα in sensing environmental cues during haploid fruiting. The wild-type MATα CPRα strain produced abundant hyphae in response to synthetic MAT a pheromone; however, the hyphal response to pheromone by Δcprα cells was significantly reduced. Exposure of wild-type cells to synthetic MAT a pheromone for 2 h induced MFα pheromone expression, whereas unexposed cells showed only basal levels of the MFα transcript. The Δcprα cells, however, exhibited only basal levels of MFα message with or without pheromone exposure, suggesting that CPRα and MFα are components of the same signaling pathway.

To understand the mechanisms leading to trastuzumab resistance in HER2-overexpressing breast tumors we created trastuzumab insensitive cell lines (SKBR3/100-8 and BT474/100-2). The cell lines maintain HER2 receptor overexpression, and show increase in EGFR. Upon trastuzumab treatment, SKBR3/100-8 and BT474/100-2 cell lines displayed increased growth rate and invasiveness. The trastuzumab resistance in SKBR3/100-8 and BT474/100-2 was accompanied with activation of the Wnt/β-catenin signaling pathway. Further investigation found that Wnt3 overexpression played a key role toward the development of trastuzumab resistance. The expression of Wnt3 in trastuzumab resistant cells increased nuclear expression of β-catenin and transactivated expression of EGFR. The increased Wnt3 in the trastuzumab resistant cells also promoted a parental EMT-like transition (epithelial to mesenchymal transition), increased N-cadherin, Twist, SLUG and decreased E-cadherin. Knockdown Wnt3 by siRNA restored cytoplasmic expression of β-catenin, and decreased EGFR expression in trastuzumab resistant cells. Furthermore the EMT markers were decreased, E-cadherin was increased and the cell invasiveness was inhibited in response to the Wnt3 down-regulation. Conversely, SKBR3 cells which had been stably transfected with full-length Wnt3 exhibited EMT-like transition. The Wnt3 transfectants, SKBR3/Wnt3-7 and SKBR3/Wnt3-9, showed significant decrease in E-cadherin and increase in N-cadherin, Twist and SLUG. The cells were less sensitive to trastuzumab compared to parental SKBR3 and vector transfected cells. In summary, our data suggests that Wnt3 overexpression activates Wnt/β-catenin signaling pathway that leads to transactivation of EGFR and promotes EMT-like transition. This could be an important mechanism leading to trastuzumab resistance in HER2 overexpressing breast cancer cells. Citation Format: Yanyuan Wu, Charles Ginther, Juri Kim, Nicole Mosher, Seyung Chung, Dennis Slamon, Jaydutt V. Vadgama. Expression of Wnt3 activates Wnt/β-catenin pathway and promotes EMT-like phenotype in trastuzumab resistant HER2-overexpressing breast cancer cells. [abstract]. In: Proceedings of the Fifth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2012 Oct 27-30; San Diego, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(10 Suppl):Abstract nr B31.

Introduction: We are interested in understanding mechanisms that lead to Trastuzumab (Herceptin) resistance in HER2 overexpressing breast tumors. In this study, we examined the role of the Wnt/β-catenin signaling pathway in Trastuzumab resistance. In addition to the Wnt pathway genes, we examined gene expression profiles using 44K gene chip array and EMT associated genes in Trastuzumab resistant cells. Methods: Wnt pathway gene profiles were examined by Agilent chip (4×44K) and Wnt/β-catenin specific array. HER2 overexpressing breast cancer cells, SKBR3 and trastuzumab resistant clone, SKBR3 100-8 cells were used as cell models. The EMT (Epithelial to Mesenchymal Transition) markers Snail, Slug, Twist and E-Cadherin were examined by both real-time PCR and western blot analysis. Cell invasiveness was determined by invasive assay. HER2-HER3 dimerization was examined using immunoprecipitation and immunoblotting procedures. Results: We established a Trastuzumab resistant cell line from the HER2 overexpressing parental SKBR3 cell line. The resistant clone called SKBR3/100-8 is insensitive to 100µg/ml of Trastuzumab. It has a mesenchymal or more basal-like morphology. It overexpresses HER2 protein and mRNA more than the parental line SKBR3. This resistant clone shows increased dimerization of HER2 with HER3. The SKBR3 100-8 cells were found to overexpress EGFR1. It is more invasive compared to wt SKBR3 and shows a significant increase in EMT marker, SLUG, but a decrease in Snail and E-cadherin compared to the wtSKBR3 cells. The resistant clone shows increase in phospho-Akt, pGSK3β and a decrease in β-Catenin. Agilent chip (4×44K) array (containing 84 Wnt pathway genes) identified the following Wnt pathway genes to be upregulated by 2 or more fold in the resistant clone: AXIN1, CSNK1A1, CSNK1D, CTBP2, DAAM1, c-JUN, TLE1, WNT3 and WNT6. Conversely, the following genes were downregulated by 2 or more fold in the resistant clone: AES, CCND3, CTNNB1, FZD1, FZD4, FZD5, PITX2, PPP2CA, PPP2R1A, WIF1, WNT11, and WNT4. The resistant clone also increased Nanog and VEGF significantly. TGFβ treatment in wtSKBR3 increased SLUG, Snail, Twist, and decreased E-Cadherin. The cells showed more mesenchymal morphology than wtSKBR without TGFβ treatment. TGFβ treatment in wtSKBR3 upregulated: c-Jun, C-Myc, CCND1, FosL1, LEF1, and TCF-1. TGFβ also induced Met, MMP1, MMP7, MMP9, MMP26, Nanog, NOS2, POU5F1, PPARd, Survivin, uPAR and VEGF. In contrast, TGFβ upregulated only c-Jun, Met, Nanog, and VEGF in SKBR3 100-8 cells. Conclusion: Wnt/β-catenin signaling is highly involved in the trastuzumab resistant breast cancer cells. The resistant signature involves c-Jun, Met, Nanog and VEGF. These results suggest a very specific signature that needs further studies for target therapy for the Trastuzumab resistant tumors and implies the use of anti VEGF therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 710. doi:10.1158/1538-7445.AM2011-710

and identified by N-terminal sequencing. MAL cDNAs were cloned and sequenced, antibodies to MAL were used to localize it in bladder urothelium by LM and EM techniques. Mice lacking the MAL gene, and those over-expressing the MAL gene, were generated by transgenic techniques. RESULTS: N-terminal sequencing of the bovine p18 identified it as the ‘myelin-and-lymphocyte-protein’ (MAL). This protein has four transmembrane domains interconnected by short hydrophilic domains. It is expressed in bladder urothelium in a differentiation-dependent manner. EM localization studies showed that it is associated with the uroplakin-delivering vesicles of all stages in urothelial umbrella cells, including the small discoidal vesicles, the mature fusiform vesicles, and the apical urothelial surface. In the latter two cases, the MAL is selectively associated with the ‘hinge areas’ that interconnect the urothelial plaques. Genetic ablation of the mouse MAL gene results in the accumulation of fusiform vesicles. Conversely, over-expression of MAL in transgenic mouse urothelium results in a reduction of the fusiform vesicles and, concurrently, the accumulation of multivesicular bodies that are involved in the endocytic degradation of uroplakins. CONCLUSIONS: Our results indicate that (i) MAL is highly expressed in bladder urothelium as a major urothelial differentiation marker, (ii) MAL knockout blocks the fusion of the fusiform vesicles with the urothelial apical surface, and (iii) MAL over-expression increases the delivery of uroplakins to apical surface and their endocytic degradation. Therefore, MAL is a key component in regulating the targeting and degradation of the uroplakin complexes, which serve as the receptor for the uropathogenic E. coli, and contribute to the bladder permeability barrier function.