Jiazhi Sun

Both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for inhibition of oncogenic K-ras prenylation but each alone is sufficient to suppress human tumor growth in nude mouse xenografts

The ability of Ras oncoproteins to cause malignant transformation requires their post-translational modifications by prenyl groups. Because K-Ras can be both farnesylated and geranylgeranylated it is not known whether both farnesyltransferase and geranylgeranyltransferase I inhibitors are required for suppressing human tumor growth in whole animals. In this paper we report that oncogenic Ras processing, MAP kinase activation and growth in nude mice are inhibited by the farnesyltransferase inhibitor FTI-276 in H- and N-Ras transformed NIH3T3 cells; whereas in KB-Ras transformed NIH3T3 cells both FTI-276 and the geranylgeranyltransferase I inhibitor GGTI-297 are required for inhibition. Furthermore, human lung A-549 and Calu-1 carcinoma cell lines were found to co-express H-, N- and K-Ras. In Calu-1 cells, the processing of H- and N-Ras is inhibited greatly by FTI-276 but only partially by GGTI-297 whereas K-Ras processing inhibition requires both FTI-276 and GGTI-297. In contrast, in A-549 cells the processing of H- and N-Ras is inhibited only by FTI-276 and K-Ras processing is resistant to co-treatment with FTI-276 and GGTI-297. Yet, the growth in nude mice of A-549 and Calu-1 xenografts, both of which express K-Ras mutations, is inhibited by FTI-276 (80% inhibition) and GGTI-297 (60%). Furthermore, FTI-276 inhibits tumor growth of NIH3T3 cells transformed by a form of oncogenic H-Ras that is exclusively geranylgeranylated and whose processing is resistant to this inhibitor. Taken together, the results demonstrate that both FTase and GGTase I inhibitors are required for inhibition of K-Ras processing but that each alone is sufficient to suppress human tumor growth in nude mice.

Cucurbitacin Q: a selective STAT3 activation inhibitor with potent antitumor activity

Constitutive activation of the JAK/STAT3 pathway is a major contributor to oncogenesis. In the present study, structure–activity relationship (SAR) studies with five cucurbitacin (Cuc) analogs, A, B, E, I, and Q, led to the discovery of Cuc Q, which inhibits the activation of STAT3 but not JAK2; Cuc A which inhibits JAK2 but not STAT3 activation; and Cuc B, E, and I, which inhibit the activation of both. Furthermore, these SAR studies demonstrated that conversion of the C3 carbonyl of the cucurbitacins to a hydroxyl results in loss of anti-JAK2 activity, whereas addition of a hydroxyl group to C11 of the cucurbitacins results in loss of anti-STAT3 activity. Cuc Q inhibits selectively the activation of STAT3 and induces apoptosis without inhibition of JAK2, Src, Akt, Erk, or JNK activation. Furthermore, Cuc Q induces apoptosis more potently in human and murine tumors that contain constitutively activated STAT3 (i.e., A549, MDA-MB-435, and v-Src/NIH 3T3) as compared to those that do not (i.e., H-Ras/NIH 3T3, MDA-MB-453, and NIH 3T3 cells). Finally, in a nude mouse tumor xenograft model, Cuc Q, but not Cuc A, suppresses tumor growth indicating that JAK2 inhibition is not sufficient to inhibit tumor growth and suggesting that the ability of Cuc Q to inhibit tumor growth is related to its anti-STAT3 activity. These studies further validate STAT3 as a drug discovery target and provide evidence that pharmacological agents that can selectively reduce the P-STAT3 levels in human cancer cells result in tumor apoptosis and growth inhibition.

The Geranylgeranyltransferase-I Inhibitor GGTI-298 Arrests Human Tumor Cells in G0/G1 and Induces p21WAF1/CIP1/SDI1 in a p53-independent Manner

Recently we have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution. Here we demonstrate that in human tumor cells the geranylgeranyltransferase-I (GGTase-I) inhibitor GGTI-298 blocked cells in G0/G1, whereas the farnesyltransferase (FTase) inhibitor FTI-277 showed a differential effect depending on the cell line. FTI-277 accumulated Calu-1 and A-549 lung carcinoma and Colo 357 pancreatic carcinoma cells in G2/M, T-24 bladder carcinoma, and HT-1080 fibrosarcoma cells in G0/G1, but had no effect on cell cycle distribution of pancreatic (Panc-1), breast (SKBr 3 and MDAMB-231), and head and neck (A-253) carcinoma cells. Furthermore, treatment of Calu-1, Panc-1, Colo 357, T-24, A-253, SKBr 3, and MDAMB-231 cells with GGTI-298, but not FTI-277, induced the protein expression levels of the cyclin-dependent kinase inhibitor p21WAF. HT-1080 and A-549 cells had a high basal level of p21WAF, and GGTI-298 did not further increase these levels. Furthermore, GGTI-298 also induces the accumulation of large amounts of p21WAF mRNA in Calu-1 cells, a cell line that lacks the tumor suppressor gene p53. There was little effect of GGTI-298 on the cellular levels of another cyclin- dependent kinase inhibitor p27KIP as well as cyclin E and cyclin D1. These results demonstrate that GGTase-I inhibitors arrest cells in G0/G1 and induce accumulation of p21WAF in a p53-independent manner and that FTase inhibitors can interfere with cell cycle events by a mechanism that involves neither p21WAF nor p27KIP. The results also point to the potential of GGTase-I inhibitors as agents capable of restoring growth arrest in cells lacking functional p53.

Akt Mediates Ras Downregulation of RhoB, a Suppressor of Transformation, Invasion, and Metastasis

ABSTRACT Although recent evidence supports a tumor-suppressive role for the GTPase RhoB, little is known about its regulation by signal transduction pathways. Here we demonstrate that Ras downregulates RhoB expression by a phosphatidylinositol 3-kinase (PI3K)- and Akt- but not Mek-dependent mechanism. Furthermore, genetic and pharmacological blockade of PI3K/Akt results in upregulation of RhoB expression. We also provide evidence for the importance of the downregulation of RhoB in oncogenesis by demonstrating that RhoB antagonizes Ras/PI3K/Akt malignancy. Ectopic expression of RhoB, but not the close relative RhoA, inhibits Ras, PI3K, and Akt induction of transformation, migration, and invasion and induces apoptosis and anoikis. Finally, RhoB inhibits melanoma metastasis to the lung in a mouse model. These studies identify suppression of RhoB as a mechanism by which the Ras/PI3K/Akt pathway induces tumor survival, transformation, invasion, and metastasis.

Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGβ (IC50 values > 100 μM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 μM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor–binding molecules with potent anticancer and antiangiogenic activity.

Inhibiting angiogenesis and tumorigenesis by a synthetic molecule that blocks binding of both VEGF and PDGF to their receptors

Angiogenesis depends on vascular endothelial growth factor (VEGF) for initiation and platelet-derived growth factor (PDGF) for maintenance of blood vessels. We have designed a targeted library of compounds from which we identified a novel molecule, GFB-204, that binds PDGF and VEGF, blocks binding of PDGF and VEGF to their receptors (200–500 nM) and subsequently inhibits PDGFR and Flk-1 tyrosine phosphorylation and stimulation of the protein kinases Erk1, Erk2 and Akt and the signal transducer and activator of transcription STAT3. GFB-204 is selective for PDGF and VEGF and does not inhibit EGF, IGF-1 and FGF stimulation of Erk1/2, Akt and STAT3. GFB-204 inhibits endothelial cell migration and capillary network formation in vitro. Finally, treatment of mice with GFB-204 suppresses human tumor growth and angiogenesis. Thus, inhibition of VEGF and PDGF receptor binding with a synthetic molecule results in potent inhibition of angiogenesis and tumorigenesis.

The BH3 α-Helical Mimic BH3-M6 Disrupts Bcl-XL, Bcl-2, and MCL-1 Protein-Protein Interactions with Bax, Bak, Bad, or Bim and Induces Apoptosis in a Bax- and Bim-dependent Manner

A critical hallmark of cancer cell survival is evasion of apoptosis. This is commonly due to overexpression of anti-apoptotic proteins such as Bcl-2, Bcl-XL, and Mcl-1, which bind to the BH3 α-helical domain of pro-apoptotic proteins such as Bax, Bak, Bad, and Bim, and inhibit their function. We designed a BH3 α-helical mimetic BH3-M6 that binds to Bcl-XL and Mcl-1 and prevents their binding to fluorescently labeled Bak- or Bim-BH3 peptides in vitro. Using several approaches, we demonstrate that BH3-M6 is a pan-Bcl-2 antagonist that inhibits the binding of Bcl-XL, Bcl-2, and Mcl-1 to multi-domain Bax or Bak, or BH3-only Bim or Bad in cell-free systems and in intact human cancer cells, freeing up pro-apoptotic proteins to induce apoptosis. BH3-M6 disruption of these protein-protein interactions is associated with cytochrome c release from mitochondria, caspase-3 activation and PARP cleavage. Using caspase inhibitors and Bax and Bak siRNAs, we demonstrate that BH3-M6-induced apoptosis is caspase- and Bax-, but not Bak-dependent. Furthermore, BH3-M6 disrupts Bcl-XL/Bim, Bcl-2/Bim, and Mcl-1/Bim protein-protein interactions and frees up Bim to induce apoptosis in human cancer cells that depend for tumor survival on the neutralization of Bim with Bcl-XL, Bcl-2, or Mcl-1. Finally, BH3-M6 sensitizes cells to apoptosis induced by the proteasome inhibitor CEP-1612.

Disruption of the Rb-Raf-1 Interaction Inhibits Tumor Growth and Angiogenesis

ABSTRACT The retinoblastoma tumor suppressor protein (Rb) plays a vital role in regulating mammalian cell cycle progression and inactivation of Rb is necessary for entry into S phase. Rb is inactivated by phosphorylation upon growth factor stimulation of quiescent cells, facilitating the transition from G1 phase to S phase. Although the signaling events after growth factor stimulation have been well characterized, it is not yet clear how these signals contact the cell cycle machinery. We had found previously that growth factor stimulation of quiescent cells lead to the direct binding of Raf-1 kinase to Rb, leading to its inactivation. Here we show that the Rb-Raf-1 interaction occurs prior to the activation of cyclin and/or cyclin-dependent kinases and facilitates normal cell cycle progression. Raf-1-mediated inactivation of Rb is independent of the mitogen-activated protein kinase cascade, as well as cyclin-dependent kinases. Binding of Raf-1 seemed to correlate with the dissociation of the chromatin remodeling protein Brg1 from Rb. Disruption of the Rb-Raf-1 interaction by a nine-amino-acid peptide inhibits Rb phosphorylation, cell proliferation, and vascular endothelial growth factor-mediated capillary tubule formation. Delivery of this peptide by a carrier molecule led to a 79% reduction in tumor volume and a 57% reduction in microvessel formation in nude mice. It appears that Raf-1 links mitogenic signaling to Rb and that disruption of this interaction could aid in controlling proliferative disorders.

Inhibition of angiogenesis by Abeta peptides.

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.

Inhibition of Angiogenesis by Aβ Peptides

Aβ peptides are naturally occurring peptides forming β-sheet aggregates that constitute an integral component of senile plaques and vascular deposits in Alzheimers disease. Since several peptides adopting a β-sheet conformation have been shown to be anti-angiogenic, we investigated the effect of Aβ on angiogenesis. We show that in vitro, Aβ dose-dependently inhibits the formation of capillaries by human brain endothelial cells plated on Matrigel and stimulates capillary degeneration at high doses. Preparations of Aβ peptides containing a higher content of β-sheet structures are more potently anti-angiogenic in vitro. Ex vivo, Aβ dose-dependently opposes angiogenesis in rat aortae and in human middle cerebral arteries. In vivo, Aβ dose dependently inhibits angiogenesis in the chick chorioallantoic membrane assay and suppresses bFGF-induced blood vessel formation in the corneal micropocket and Matrigel plug assays. Since angiogenesis is required for tumor growth, we explored the effect of Aβ on human glioblastoma (U87MG) and human lung adenocarcinoma (A549) tumors. We show that intra-tumoral injection of Aβ potently inhibits the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumor xenografts in nude mice. Similarly to the intra-tumoral injection regimen, Aβ delivered intraperitoneally also suppressed the growth of human lung adenocarcinoma tumor xenografts. Altogether our data show that Aβ is an angiogenesis inhibitor.