Date of Graduation

5-2010

Program Affiliation

Cancer Biology

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Keping Xie

Committee Member

Kenneth Aldape

Committee Member

Zhen Fan

Committee Member

Juan Fueyo-Margareto

Committee Member

Suyun Huang

Abstract

Transcription factor Specificity Protein 1(Sp1) is reported to be essential for vascular endothelial growth factor (VEGF) constitutive expression in human pancreatic adenocarcinoma. The definitive role of Sp1 in angiogenesis, the impact of anti-angiogenic therapy on the Sp1/ VEGF signaling and the Sp1 signaling alteration on the anti-angiogenic therapy effect are unclear. The understanding of Sp1 regulation on VEGF and their interactions has significant clinical implications.

Sp1 and VEGF expression and microvessel density (MVD) were analyzed using pancreatic cancer patients specimens through immunohistochemistry staining. The impact of Sp1 expression alternation on angiogenesis and tumor progression in nude mice were determined by knockdown Sp1 with small-interfering RNA (siRNA). Sp1 protein expression was correlated with the MVD (P < 0.001) and VEGF expression (P < 0.05). In mouse models, tumor progression and metastasis were inhibited after knockdown of Sp1 expression. The antitumor activity was correlated with the down-regulation of Sp1 downstream angiogenic factors caused by Sp1 knockdown.

Sp1 and its downstream angiogenic genes expression were suppressed by mithramycin treatment both in vitro and in vivo. Moreover, mithramycin treatment reduced MVD in vivo. This was consistent with the down-regulation of VEGF, PDGF, and EGFR. Human xenograft pancreatic tumor growth was suppressed by Bevacizumab treatment. Both western blot and immunohistochemistry staining revealed that Sp1 and its downstream angiogenic genes expression were up-regulated by Bevacizumab treatment. Bevacizumab and mithramycin combination treatment synergistically suppressed tumor growth in vivo. This is correlated with the down-regulation of Sp1 and its downstream angiogenic genes expression. Bevacizumab treatment may trigger a positive feedback to up-regulate angiogenic factors through Sp1 trans-activation and this mechanism can be diminished by mithramycin treatment.

Combination treatment of mithramycin and tolfenamic acid which has been shown to facilitate Sp1 protein degradation had synergistic cell growth inhibition effect in vitro. In vivo, metronomic low-dose combined treatment of mithramycin and tolfenamic acid produced tumor suppression in mouse model. Gene expression analysis showed that the combination treatment synergistic down-regulated Sp1 and its downstream angiogenic molecule VEGF.

In summary, experimental results and clinical research suggested that Sp1 signaling is very important for angiogenesis of pancreatic cancer. Mithramycin treatment down-regulated Sp1 protein through interpose its transcription and decreased Sp1 downstream angiogenic genes. These down-regulations were correlated with the antitumor activity. Synergistic down-regulation of Sp1 and decreased expression of its downstream angiogenic molecules in turn significant reduced the angiogenic potential of pancreatic cancer cells and is an effective anti-angiogenesis strategy. Therefore, this study showed that Sp1 is a key factor of angiogenesis and manipulation of Sp1/VEGF signaling has clinical implication in anti-angiogenic therapy of pancreatic cancer.

Keywords

Sp1, Pancreatic Cancer, Angiogenesis, Mithramycin