Targeting the eIF4A RNA helicase blocks translation of the MUC1-C oncoprotein

The oncogenic MUC1-C subunit is aberrantly overexpressed in most human breast cancers by mechanisms that are not well understood. The present studies demonstrate that stimulation of non- malignant MCF-10A cells with epidermal growth factor (EGF) or heregulin (HRG) results in marked upregulation of MUC1-C translation. Growth factor-induced MUC1-C translation was found to be mediated by PI3K->AKT, and not MEK->ERK1/2, signaling. We also show that activation of the mTORC1->S6K1 pathway decreases PDCD4, an inhibitor of the eIF4A RNA helicase, and contributes to the induction of MUC1-C translation. In concert with these results, treatment of growth factor-stimulated MCF-10A cells with the eIF4A RNA helicase inhibitors, silvestrol and CR-1-31-B, blocked increases in MUC1-C abundance. The functional significance of the increase in MUC1-C translation is supported by the demonstration that MUC1-C, in turn, forms complexes with EGFR and promotes EGFR-mediated activation of the PI3K->AKT pathway and the induction of growth. Compared to MCF-10A cells, constitutive overexpression of MUC1-C in breast cancer cells was unaffected by EGF stimulation, but was blocked by inhibiting PI3K->AKT signaling. The overexpression of MUC1-C in breast cancer cells was also inhibited by blocking eIF4A RNA helicase activity with silvestrol and CR-1-31-B. These findings indicate that EGF-induced MUC1-C expression is mediated by the PI3K->AKT pathway and the eIF4A RNA helicase, and that this response promotes EGFR signaling in an autoinductive loop. The findings also indicate that targeting the eIF4A RNA helicase is a novel approach for blocking MUC1-C overexpression in breast cancer cells.P50 CA100707 - NCI NIH HHS; R01 CA042802 - NCI NIH HHS; R01 CA097098 - NCI NIH HHS; R01 GM073855 - NIGMS NIH HH

MUC1-C drives myeloid leukaemogenesis and resistance to treatment by a survivin-mediated mechanism

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy with an unmet need for improved therapies. Responses to standard cytotoxic therapy in AML are often transient because of the emergence of chemotherapy-resistant disease. The MUC1-C oncoprotein governs critical pathways of tumorigenesis, including self-renewal and survival, and is aberrantly expressed in AML blasts and leukaemia stem cells (LSCs). However, a role for MUC1-C in linking leukaemogenesis and resistance to treatment has not been described. In this study, we demonstrate that MUC1-C overexpression is associated with increased leukaemia initiating capacity in an NSG mouse model. In concert with those results, MUC1-C silencing in multiple AML cell lines significantly reduced the establishment of AML in vivo. In addition, targeting MUC1-C with silencing or pharmacologic inhibition with GO-203 led to a decrease in active β-catenin levels and, in-turn, down-regulation of survivin, a critical mediator of leukaemia cell survival. Targeting MUC1-C was also associated with increased sensitivity of AML cells to Cytarabine (Ara-C) treatment by a survivin-dependent mechanism. Notably, low MUC1 and survivin gene expression were associated with better clinical outcomes in patients with AML. These findings emphasize the importance of MUC1-C to myeloid leukaemogenesis and resistance to treatment by driving survivin expression. Our findings also highlight the potential translational relevance of combining GO-203 with Ara-C for the treatment of patients with AML

MUC1-C confers EMT and KRAS independence in mutant KRAS lung cancer cells

Non-small cell lung cancers (NSCLCs) that harbor an oncogenic KRAS mutation are often associated with resistance to targeted therapies. The MUC1-C transmembrane protein is aberrantly overexpressed in NSCLCs and confers a poor outcome; however, the functional role for MUC1-C in mutant KRAS NSCLC cells has remained unclear. The present studies demonstrate that silencing MUC1-C in A549/KRAS(G12S) and H460/KRAS(Q61H) NSCLC cells is associated with downregulation of AKT signaling and inhibition of growth. Overexpression of a MUC1-C(CQC→AQA) mutant, which inhibits MUC1-C homodimerization and function, suppressed both AKT and MEK activation. Moreover, treatment with GO-203, an inhibitor of MUC1-C homodimerization, blocked AKT and MEK signaling and decreased cell survival. The results further demonstrate that targeting MUC1-C suppresses expression of the ZEB1 transcriptional repressor by an AKT-mediated mechanism, and in turn induces miR-200c. In concert with these effects on the ZEB1/miR-200c regulatory loop, targeting MUC1-C was associated with reversal of the epithelial-mesenchymal transition (EMT) and inhibition of self-renewal capacity. Loss of MUC1-C function also attenuated KRAS independence and inhibited growth of KRAS mutant NSCLC cells as tumors in mice. These findings support a model in which targeting MUC1-C inhibits mutant KRAS signaling in NSCLC cells and thereby reverses the EMT phenotype and decreases self-renewal

Survivin a radiogenetic promoter for glioblastoma viral gene therapy independently from CArG motifs

BACKGROUND: Radiogenetic therapy is a novel approach in the treatment of cancer, which employs genetic modification to alter the sensitivity of tumor cells to the effect of applied radiation. AIM: To select a potent radiation inducible promoter in the context of brain tumors and to investigate if CArG radio responsive motifs or other elements in the promoter nucleotide sequences can correlate to its response to radiation. METHODS: To select initial candidates for promoter inducible elements, the levels of mRNA expression of six different promoters were assessed using Quantitative RTPCR in D54 MG cells before and after radiation exposure. Recombinant Ad/reporter genes driven by five different promoters; CMV, VEGF, FLT-1, DR5 and survivin were constructed. Glioma cell lines were infected with different multiplicity of infection of the (promoter) Ad or CMV Ad. Cells were then exposed to a range of radiation (0–12 Gy) at single fraction. Fluorescent microscopy, Luc assay and X-gal staining was used to detect the level of expression of related genes. Different glioma cell lines and normal astrocytes were infected with Ad survivin and exposed to radiation. The promoters were analyzed for presence of CArG radio-responsive motifs and CCAAT box consensus using NCBI blast bioinformatics software. RESULTS: Radiotherapy increases the expression of gene expression by 1.25–2.5 fold in different promoters other than survivin after 2 h of radiation. RNA analysis was done and has shown an increase in copy number of tenfold for survivin. Most importantly cells treated with RT and Ad Luc driven by survivin promoter showed a fivefold increase in expression after 2 Gy of radiation in comparison to non-irradiated cells. Presence or absence of CArG motifs did not correlate with promoter response to radiation. Survivin with the best response to radiation had the lowest number of CCAAT box. CONCLUSION: Survivin is a selective potent radiation inducible promoter for glioblastoma viral gene therapy and this response to radiation could be independent of CArG motifs

Targeting the MUC1-C oncoprotein inhibits self-renewal capacity of breast cancer cells

The capacity of breast cancer cells to form mammospheres in non-adherent serum-free culture is used as a functional characteristic of the self-renewing stem-like cell population. The present studies demonstrate that silencing expression of the MUC1-C oncoprotein inhibits growth of luminal MCF-7 and HER2-overexpressing SKBR3 breast cancer cells as mammospheres. We also show that triple-negative MDA-MB-468 breast cancer cells are dependent on MUC1-C for growth as mammospheres and tumor xenografts. Similar results were obtained when MUC1-C function was inhibited by expression of a MUC1-C(CQC→AQA) mutant. Moreover, treatment with the MUC1-C inhibitor GO-203, a cell penetrating peptide that binds to the MUC1-C cytoplasmic domain and blocks MUC1-C function, confirmed the importance of this target for self-renewal. The mechanistic basis for these findings is supported by the demonstration that MUC1-C activates NF-κB, occupies the IL-8 promoter with NF-κB, and induces IL-8 transcription. MUC1-C also induces NF-κB-dependent expression of the IL-8 receptor, CXCR1. In concert with these results, targeting MUC1-C with GO-203 suppresses IL-8/CXCR1 expression and disrupts the formation of established mammospheres. Our findings indicate that MUC1-C contributes to the self-renewal of breast cancer cells by activating the NF-κB→IL-8/CXCR1 pathway and that targeting MUC1-C represents a potential approach for the treatment of this population

TARGETING THE MUC1-C ONCOPROTEIN DOWNREGULATES HER2 ACTIVATION AND ABROGATES TRASTUZUMAB RESISTANCE IN BREAST CANCER CELLS

Patients with HER2 positive breast cancer often exhibit intrinsic or acquired resistance to trastuzumab treatment. The transmembrane MUC1-C oncoprotein is aberrantly overexpressed in breast cancer cells and associates with HER2. The present studies demonstrate that silencing MUC1-C in HER2-overexpressing SKBR3 and BT474 breast cancer cells results in downregulation of constitutive HER2 activation. Moreover, treatment with the MUC1-C inhibitor, GO-203, was associated with disruption of MUC1-C/HER2 complexes and decreases in tyrosine phosphorylated HER2 (p-HER2) levels. In studies of trastuzumab-resistant SKBR3R and BT474R cells, we found that the association between MUC1-C and HER2 is markedly increased (~20-fold) as compared to that in sensitive cells. Additionally, silencing MUC1-C in the trastuzumab-resistant cells or treatment with GO-203 decreased p-HER2 and AKT activation. Moreover, targeting MUC1-C was associated with downregulation of phospho-p27 and cyclin E, which confer trastuzumab resistance. Consistent with these results, targeting MUC1-C inhibited the growth and clonogenic survival of both trastuzumab-resistant cells. Our results further demonstrate that silencing MUC1-C reverses resistance to trastuzumab and that the combination of GO-203 and trastuzumab is highly synergistic. These findings indicate that MUC1-C contributes to constitutive activation of the HER2 pathway and that targeting MUC1-C represents a potential approach to abrogate trastuzumab resistance

Cooperative Interaction between the MUC1-C Oncoprotein and the Rab31 GTPase in Estrogen Receptor-Positive Breast Cancer Cells

Rab31 is a member of the Ras superfamily of small GTPases that has been linked to poor outcomes in patients with breast cancer. The MUC1-C oncoprotein is aberrantly overexpressed in most human breast cancers and also confers a poor prognosis. The present results demonstrate that MUC1-C induces Rab31 expression in estrogen receptor positive (ER+) breast cancer cells. We show that MUC1-C forms a complex with estrogen receptor α (ERα) on the Rab31 promoter and activates Rab31 gene transcription in an estrogen-dependent manner. In turn, Rab31 contributes to the upregulation of MUC1-C abundance in breast cancer cells by attenuating degradation of MUC1-C in lysosomes. Expression of an inactive Rab31(S20N) mutant in nonmalignant breast epithelial cells confirmed that Rab31 regulates MUC1-C expression. The functional significance of the MUC1-C/Rab31 interaction is supported by the demonstration that Rab31 confers the formation of mammospheres by a MUC1-C-dependent mechanism. Analysis of microarray databases further showed that (i) Rab31 is expressed at higher levels in breast cancers as compared to that in normal breast tissues, (ii) MUC1+ and ER+ breast cancers have increased levels of Rab31 expression, and (iii) patients with Rab31-positive breast tumors have a significantly decreased ten-year overall survival as compared to those with Rab31-negative tumors. These findings indicate that MUC1-C and Rab31 function in an autoinductive loop that contributes to overexpression of MUC1-C in breast cancer cells

Cockayne Syndrome Protein B Interacts with and Is Phosphorylated by c-Abl Tyrosine Kinase

The Cockayne Syndrome group B (CSB) protein plays important roles in transcription, transcription-coupled nucleotide excision repair and base excision DNA repair. c-Abl kinase also plays a role in DNA repair as a regulator/coordinator of the DNA damage response. This study presents evidence that the N-terminal region of CSB interacts with the SH3 domain of c-Abl in vitro and in vivo. In addition, c-Abl kinase phosphorylates CSB at Tyr932. The subcellular localization of CSB to the nucleus and nucleolus is altered after phosphorylation by c-Abl. c-Abl-dependent phosphorylation of CSB increased in cells treated with hydrogen peroxide and decreased in cells pre-treated with STI-571, a c-Abl-specific protein kinase inhibitor. Activation of the c-Abl kinase in response to oxidative damage is not observed in CSB null cells. These results suggest that c-Abl and CSB may regulate each other in a reciprocal manner in response to oxidative stress