Laffin, Brian Edward (2008-08). Regulation of epithelial-mesenchymal transition and DNA damage responses by singleminded-2s. Doctoral Dissertation. Thesis uri icon

abstract

  • Virtually all signaling pathways that play key roles in development such as the transfroming growth factor (TGF)-beta, notch, and wnt pathways also influence tumor formation, implying that cancer is in a sense development gone awry. Therefore, identification and elucidation of developmental pathways has great potential for generating new diagnostic tools and molecular therapy targets. Singleminded-2s (SIM2s), a splice variant of the basic helilx-loop-helix / PER-ARNT-SIM (bHLH/PAS) transcriptional repressor Singleminded-2, is lost or repressed in approximately 70% of human breast tumors and has a profound influence on normal mammary development. In order to gain a better understanding of the mechanisms by which SIM2s restricts malignant transformation and progression in breast cancer, we depleted SIM2 RNA in MCF-7 cells using a retroviral shRNA system and examined gene expression and functional abilities of the SIM2-depleted MCF-7 cells (SIM2i) relative to a control MCF line expressing a non-specific "scrambled" shRNA (SCR). Depletion of SIM2 resulted in an epithelial-mesenchymal transition (EMT)-like effect characterized by increased migration and invasion, altered morphology, and loss of epithelial markers concomitant with gain of mesenchymal markers. The root of this effect may be loss of SIM2- mediated repression of the E-cadherin repressor slug, as SIM2 is able to bind and repress transcription from the slug promoter, and slug expression is dramatically elevated in SIM2i MCF-7 cells. Consistent with the previously established role of slug in resistance to various cancer therapies, SIM2i cells are resistant to the radiomimetic doxorubicin and appear to have elevated self-renewal capacity under certain conditions. Intriguingly, SIM2 protein levels are elevated by treatment with DNA damaging agents, and SIM2 interacts with the p53 complex via co-regulation of specific p53- target gene such as p21/WAF1/CIP1. These results provide a plausible mechanism for the tumor suppressor activity of SIM2, and provide insight into a novel tumor suppressive transcriptional circuit that may have utility as a therapeutic target.
  • Virtually all signaling pathways that play key roles in development such as the
    transfroming growth factor (TGF)-beta, notch, and wnt pathways also influence tumor
    formation, implying that cancer is in a sense development gone awry. Therefore,
    identification and elucidation of developmental pathways has great potential for
    generating new diagnostic tools and molecular therapy targets. Singleminded-2s
    (SIM2s), a splice variant of the basic helilx-loop-helix / PER-ARNT-SIM (bHLH/PAS)
    transcriptional repressor Singleminded-2, is lost or repressed in approximately 70% of
    human breast tumors and has a profound influence on normal mammary development. In
    order to gain a better understanding of the mechanisms by which SIM2s restricts
    malignant transformation and progression in breast cancer, we depleted SIM2 RNA in
    MCF-7 cells using a retroviral shRNA system and examined gene expression and
    functional abilities of the SIM2-depleted MCF-7 cells (SIM2i) relative to a control MCF
    line expressing a non-specific "scrambled" shRNA (SCR). Depletion of SIM2 resulted
    in an epithelial-mesenchymal transition (EMT)-like effect characterized by increased migration and invasion, altered morphology, and loss of epithelial markers concomitant
    with gain of mesenchymal markers. The root of this effect may be loss of SIM2-
    mediated repression of the E-cadherin repressor slug, as SIM2 is able to bind and repress
    transcription from the slug promoter, and slug expression is dramatically elevated in
    SIM2i MCF-7 cells. Consistent with the previously established role of slug in resistance
    to various cancer therapies, SIM2i cells are resistant to the radiomimetic doxorubicin
    and appear to have elevated self-renewal capacity under certain conditions. Intriguingly,
    SIM2 protein levels are elevated by treatment with DNA damaging agents, and SIM2
    interacts with the p53 complex via co-regulation of specific p53- target gene such as
    p21/WAF1/CIP1. These results provide a plausible mechanism for the tumor suppressor
    activity of SIM2, and provide insight into a novel tumor suppressive transcriptional
    circuit that may have utility as a therapeutic target.

publication date

  • August 2008