Dentin sialoprotein (DSP) and phosphophoryn (PP), acidic proteins critical to dentin mineralization, are translated from a single transcript as a DSP-PP precursor that undergoes specific proteolytic processing to generate DSP and PP. The cleavage mechanism continues to be controversial, in part because of the difficulty of obtaining DSP-PP from mammalian cells and dentin matrix. We have infected Sf9 cells with a recombinant baculovirus to produce large amounts of secreted DSP-PP240, a variant form of rat DSP-PP. Mass spectrometric analysis shows that DSP-PP240 secreted by Sf9 cells undergoes specific cleavage at the site predicted from the N-terminal sequence of PP extracted from dentin matrix: SMQG447↓D448DPN. DSP-PP240 is cleaved after secretion by a zinc-dependent activity secreted by Sf9 cells, generating DSP430 and PP240 products that are stable in the medium. DSP-PP processing activity is constitutively secreted by Sf9 cells, but secretion is diminished 3 days after infection. Using primers corresponding to the highly conserved catalytic domain of Drosophila melanogaster tolloid (a mammalian BMP1 homolog), we isolated a partial cDNA for a Spodopotera frugiperda tolloid-related-1 protein (TLR1) that is 78% identical to Drosophila TLR1 but only 65% identical to Drosophila tolloid. Tlr1 mRNA decreased rapidly in Sf9 cells after baculovirus infection and was undetectable 4d after infection, paralleling the observed decrease in secretion of the DSP-PP240 processing activity after infection. Human BMP1 is more similar to Sf9 and Drosophila TLR1 than to tolloid, and Sf9 TLR1 is more similar to BMP1 than to other mammalian homologs. Recombinant human BMP1 correctly processed baculovirus-expressed DSP-PP240 in a dose-dependent manner. Together, these data suggest that the physiologically accurate cleavage of mammalian DSP-PP240 in the Sf9 cell system represents the action of a conserved processing enzyme and support the proposed role of BMP1 in processing DSP-PP in dentin matrix.

Recent studies have demonstrated that cancer stem cells play an important role in the pathobiology of head and neck squamous cell carcinomas (HNSCC). However, little is known about functional interactions between head and neck cancer stem-like cells (CSC) and surrounding stromal cells. Here, we used Aldehyde Dehydrogenase activity and CD44 expression to sort putative stem cells from primary human HNSCC. Implantation of 1,000 CSC (ALDH+CD44+Lin−) led to tumors in 13 (out of 15) mice, while 10,000 non-cancer stem cells (NCSC; ALDH−CD44−Lin−) resulted in 2 tumors in 15 mice. These data demonstrated that ALDH and CD44 select a sub-population of cells that are highly tumorigenic. The ability to self-renew was confirmed by the observation that ALDH+CD44+Lin− cells sorted from human HNSCC formed more spheroids (orospheres) in 3-D agarose matrices or ultra-low attachment plates than controls and were serially passaged in vivo. We observed that approximately 80% of the CSC were located in close proximity (within 100-µm radius) of blood vessels in human tumors, suggesting the existence of perivascular niches in HNSCC. In vitro studies demonstrated that endothelial cell-secreted factors promoted self-renewal of CSC, as demonstrated by the upregulation of Bmi-1 expression and the increase in the number of orospheres as compared to controls. Notably, selective ablation of tumor-associated endothelial cells stably transduced with a caspase-based artificial death switch (iCaspase-9) caused a marked reduction in the fraction of CSC in xenograft tumors. Collectively, these findings indicate that endothelial cell-initiated signaling can enhance the survival and self-renewal of head and neck cancer stem cells.

Purpose

To investigate the effect of a metronomic (low dose, high frequency) small molecule inhibitor of Bcl-2 (TW-37) in combination with radiotherapy on microvascular endothelial cells in vitro and in tumor angiogenesis in vivo.

Methods and materials

Primary human dermal microvascular endothelial cells (HDMEC) were exposed to ionizing radiation and/or TW-37, and colony formation as well as capillary sprouting in 3-D collagen matrices, was evaluated. Xenografts vascularized with human blood vessels were engineered by co-transplantation of human squamous cell carcinoma cells (OSCC3) and HDMEC seeded in highly porous biodegradable scaffolds into the subcutaneous space of immunodeficient mice. Mice were treated with metronomic TW-37 and/or radiation, and tumor growth was evaluated.

Results

Low dose TW-37 sensitized primary endothelial cells to radiation-induced inhibition of colony formation. Low dose TW-37 or radiation partially inhibited endothelial cell sprout formation, while in combination these therapies abrogated new sprouting. Combination of metronomic TW-37 and low dose radiation inhibited tumor growth and resulted in significant increase in time to failure as compared to controls, whereas single agents did not. Notably, histopathological analysis revealed that tumors treated with TW-37 (with or without radiation) are more differentiated and showed more cohesive invasive fronts, which is consistent with less aggressive phenotype.

Conclusions

These results demonstrate that metronomic TW-37 potentiates the anti-tumor effects of radiotherapy, and suggest that patients with head and neck cancer might benefit from the combination of small molecule inhibitor of Bcl-2 and radiation therapy.

It is well known that angiogenesis plays a critical role in the pathobiology of tumors. Recent clinical trials have shown that inhibition of angiogenesis can be an effective therapeutic strategy for patients with cancer. However, one of the outstanding issues in anti-angiogenic treatment for cancer is the development of toxicities related to off-target effects of drugs. Transcriptional targeting of tumor endothelial cells involves the use of specific promoters for selective expression of therapeutic genes in the endothelial cells lining the blood vessels of tumors. Recently, several genes that are expressed specifically in tumor-associated endothelial cells have been identified and characterized. These discoveries have enhanced the prospectus of transcriptionaly targeting tumor endothelial cells for cancer gene therapy. In this manuscript, we review the promoters, vectors, and therapeutic genes that have been used for transcriptional targeting of tumor endothelial cells, and discuss the prospects of such approaches for cancer gene therapy.

Antiangiogenic therapies have shown varying results partly because each tumor type secretes a distinct panel of angiogenic factors to sustain its own microvascular network. In addition, recent evidence demonstrated that tumors develop resistance to antiangiogenic therapy by turning on alternate angiogenic pathways when one pathway is therapeutically inhibited. Here, we test the hypothesis that expression of a caspase-based artificial death switch in tumor-associated endothelial cells will disrupt tumor blood vessels and slowdown tumor progression irrespective of tumor type. Adenoviral vectors expressing inducible Caspase-9 (iCaspase-9) under transcriptional regulation with the endothelial cell specific VEGFR2 promoter (Ad-hVEGFR2-iCaspase-9) induced apoptosis of proliferating human dermal microvascular endothelial cells (HDMEC), but not human tumor cells (UM-SCC-17B, head and neck squamous cell carcinoma; HepG2, hepatocellular carcinoma; PC-3, prostate adenocarcinoma; SLK, Kaposi’s sarcoma; MCF-7, breast adenocarcinoma). Notably, apoptosis was dependent upon activation of iCaspase-9 with the dimerizer drug AP20187. Local delivery of Ad-hVEGFR2-iCaspase-9 followed by intraperitoneal injection of AP20187 ablated tumor microvessels and inhibited xenografted tumor growth in all tumor models evaluated here. We conclude that a cancer gene therapy strategy based on a transcriptionally targeted viral vector expressing an inducible caspase allows for selective and controlled ablation of microvessels of histopathologically diverse tumor types.

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF165) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.

Phenotype analysis of female mice lacking androgen receptor (AR) deficient (AR−/−) indicates that the development of mammary glands is retarded with reduced ductal branching in the prepubertal stages, and fewer Cap cells in the terminal end buds, as well as decreased lobuloalveolar development in adult females, and fewer milk-producing alveoli in the lactating glands. The defective development of AR−/− mammary glands involves the defects of insulin-like growth factor I–insulin-like growth factor I receptor and mitogen-activated protein kinase (MAPK) signals as well as estrogen receptor (ER) activity. Similar growth retardation and defects in growth factor–mediated Ras/Raf/MAPK cascade and ER signaling are also found in AR−/− MCF7 breast cancer cells. The restoration assays show that AR NH2-terminal/DNA-binding domain, but not the ligand-binding domain, is essential for normal MAPK function in MCF7 cells, and an AR mutant (R608K), found in male breast cancer, is associated with the excessive activation of MAPK. Together, our data provide the first in vivo evidence showing that AR-mediated MAPK and ER activation may play important roles for mammary gland development and MCF7 breast cancer cell proliferation.