Clones of Tumor Cells Derived from a Single Primary Human Lung Tumor Reveal Different Patterns of β1 Integrin Expression

Previously we reported that over 75% of human non-small cell lung cancers overexpress the βi integrin VLA-2 on their surface and show an increase in the mRNA encoding the α-2 chain of this integrin. These results suggested the possibility that the overproduction and overexpression of one or more of the β₁ integrin may be involved in the pathogenesis of human lung tumors by modulating the invasive and/or metastatic potential of the tumor. We report here the generation and characterization of multiple clones of tumor cells derived from the primary culture of cells obtained from biopsy tissue of an aggressive human squamous cell lung tumor. We show that these tumor clones (or clonotypes) exhibit seven different yet stable phenotypes with respect to the expression of five members of the βi integrin family. These results illustrate that a primary human lung tumor consists of multiple subpopulations of cells that while indistinguishable by ultrastructure are heterogeneous with respect to their β₁ integrins. The availability of these distinct tumor clonotypes derived from a single tumor biopsy have made it possible to test the assumption that the βi integrins play a role in tumor progression. The feasibility of this approach is demonstrated here by the intravenous inoculation of different human tumor clonotypes into severe combined immunodeficient (scid) mice. Our preliminary results with a pair of tumor clonotypes differing in VLA-1 and VLA-2 expression level reveal that the clonotype with high level of VLA-1 and VLA-2 displays a substantial increase in the experimental engraftment and metastasis of the human tumor cells in scid mice.     

Hereditary Hyperparathyroidism–Jaw Tumor Syndrome: The Endocrine Tumor Gene HRPT2 Maps to Chromosome 1q21-q31

The syndrome of hereditary hyperparathyroidism and jaw tumors (HPT-JT) is characterized by inheritance, in an autosomal dominant pattern, of recurrent parathyroid adenomas, fibro-osseous tumors of the mandible and/or maxilla, Wilms tumor, and parathyroid carcinoma. This syndrome is clinically and genetically distinct from other endocrine neoplasia syndromes and appears to result from mutation of an endocrine tumor gene designated “HRPT2.” We studied five HPT-JT families (59 persons, 20 affected); using PCR-based markers, we instituted a genomewide linkage search after excluding several candidate genes. Lod scores were calculated at various recombination fractions (θ), penetrance 90%. We mapped HRPT2 to the long arm of chromosome 1 (1q21-q31). The maximal lod score was 6.10 at θ = .0 with marker D1S212, or >10⁶ odds in favor of linkage. In six hereditary Wilms tumor families (96 persons, 29 affected), we found no linkage to 1q markers closely linked with HRPT2 (lod scores ?15.6 [D1S191] and ?17.8 [D1S196], θ = .001). Nine parathyroid adenomas and one Wilms tumor from nine members of three HPT-JT families were examined for loss of heterozygosity at linked loci. The parathyroid adenomas and Wilms tumor showed no loss of heterozygosity for these DNA markers. Our data establish that HRPT2, an endocrine tumor gene on the long arm of chromosome 1, is responsible for the HPT-JT syndrome but not for the classical hereditary Wilms tumor syndrome.     

Tumor Associated Macrophages Protect Colon Cancer Cells from TRAIL-Induced Apoptosis through IL-1β- Dependent Stabilization of Snail in Tumor Cells

Background

We recently reported that colon tumor cells stimulate macrophages to release IL-1β, which in turn inactivates GSK3β and enhances Wnt signaling in colon cancer cells, generating a self-amplifying loop that promotes the growth of tumor cells.

Principal Findings

Here we describe that macrophages protect HCT116 and Hke-3 colon cancer cells from TRAIL-induced apoptosis. Inactivation of IL-1β by neutralizing IL-1β antibody, or silencing of IL-1β in macrophages inhibited their ability to counter TRAIL-induced apoptosis. Accordingly, IL-1β was sufficient to inhibit TRAIL-induced apoptosis. TRAIL-induced collapse of the mitochondrial membrane potential (Δψ) and activation of caspases were prevented by macrophages or by recombinant IL-1β. Pharmacological inhibition of IL-1β release from macrophages by vitamin D₃, a potent chemopreventive agent for colorectal cancer, restored the ability of TRAIL to induce apoptosis of tumor cells cultured with macrophages. Macrophages and IL-1β failed to inhibit TRAIL-induced apoptosis in HCT116 cells expressing dnIκB, dnAKT or dnTCF4, confirming that they oppose TRAIL-induced cell death through induction of Wnt signaling in tumor cells. We showed that macrophages and IL-1β stabilized Snail in tumor cells in an NF-κB/Wnt dependent manner and that Snail deficient tumor cells were not protected from TRAIL-induced apoptosis by macrophages or by IL-1β, demonstrating a crucial role of Snail in the resistance of tumor cells to TRAIL.

Significance

We have identified a positive feedback loop between tumor cells and macrophages that propagates the growth and promotes the survival of colon cancer cells: tumor cells stimulate macrophages to secrete IL-1β, which in turn, promotes Wnt signaling and stabilizes Snail in tumor cells, conferring resistance to TRAIL. Vitamin D₃ halts this amplifying loop by interfering with the release of IL-1β from macrophages. Accordingly, vitamin D₃ sensitizes tumor cells to TRAIL-induced apoptosis, suggesting that the therapeutic efficacy of TRAIL could be augmented by this readily available chemopreventive agent.     

HIF1α Suppresses Tumor Cell Proliferation through Inhibition of Aspartate Biosynthesis

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Ultrasound Targeted Apoptosis Imaging in Monitoring Early Tumor Response of Trastuzumab in a Murine Tumor Xenograft Model of Her-2–Positive Breast Cancer1

OBJECTIVE: Our study aimed to monitor the trastuzumab therapy response of murine tumor xenograft model with human epidermal growth factor receptor 2 (Her-2)–positive breast cancer using ultrasound targeted apoptosis imaging. METHODS: We prepared targeted apoptosis ultrasound probes by nanobubble (NB) binding with Annexin V. In vitro, we investigated the binding rate of NB–Annexin V with breast cancer apoptotic cells after the trastuzumab treatment. In vivo, tumor-bearing mice underwent ultrasound targeted imaging over 7 days. After imaging was completed, the tumors were excised to determine Her-2 and caspase-3 expression by immunohistochemistry (IHC). The correlation between parameters of imaging and histologic results was then analyzed. RESULTS: For seeking the ability of targeted NB binding with apoptotic tumor cells (Her-2 positive), we found that binding rate in the treatment group was higher than that of the control group in vitro (P = .001). There were no differences of tumor sizes in all groups over the treatment process in vivo (P = .98). However, when using ultrasound imaging to visualize tumors by targeted NB in vivo, we observed that the mean and peak intensities from NBs gradually increased in the treatment group after trastuzumab therapy (P = .001). Furthermore, these two parameters were significantly associated with caspase-3 expression of tumor excised samples (P = .0001). CONCLUSION: Ultrasound targeted apoptosis imaging can be a non-invasive technique to evaluate the early breast tumor response to trastuzumab therapy.     

Tumor necrosis factor-α stimulation endothelial-to-mesenchymal transition during cardiac fibrosis via endothelin-1 signaling

Cardiac fibrosis is an important pathological change after myocardial infarction (MI). High concentration of tumor necrosis factor-α (TNF-α) contributes to cardiac fibrosis, and TNF-α has been demonstrated to be involved in transforming growth factor-β1-induced endothelial-to-mesenchymal transition (EndMT). However, the role and molecular mechanisms of TNF-α during cardiac fibrosis remain largely unexplored. In this study, we demonstrated that TNF-α and endothelin-1 (ET-1) were upregulated in cardiac fibrosis after MI, and genes associated with EndMT were also upregulated. An in vitro model of EndMT demonstrated that TNF-α promoted EndMT by upregulation of vimentin and α-smooth muscle actin, and which strongly increased ET-1 expression. ET-1 promoted TNF-α-induced expression of gene program through phosphorylation levels of SMAD family member 2, while subsequent inhibition of ET-1 almost abolished the effect of TNF-α during the process of EndMT. In summary, these findings demonstrated that ET-1 is involved in the EndMT induced by TNF-α during cardiac fibrosis.     

β-Arrestin2 Regulates Lysophosphatidic Acid-Induced Human Breast Tumor Cell Migration and Invasion via Rap1 and IQGAP1

β-arrestins play critical roles in chemotaxis and cytoskeletal reorganization downstream of several receptor types, including G protein-coupled receptors (GPCRs), which are targets for greater than 50% of all pharmaceuticals. Among them, receptors for lysophosphatidic acid (LPA), namely LPA₁ are overexpressed in breast cancer and promote metastatic spread. We have recently reported that β-arrestin2 regulates LPA₁-mediated breast cancer cell migration and invasion, although the underlying molecular mechanisms are not clearly understood. We show here that LPA induces activity of the small G protein, Rap1 in breast cancer cells in a β-arrestin2-dependent manner, but fails to activate Rap1 in non-malignant mammary epithelial cells. We found that Rap1A mRNA levels are higher in human breast tumors compared to healthy patient samples and Rap1A is robustly expressed in human ductal carcinoma in situ and invasive tumors, in contrast to the normal mammary ducts. Rap1A protein expression is also higher in aggressive breast cancer cells (MDA-MB-231 and Hs578t) relative to the weakly invasive MCF-7 cells or non-malignant MCF10A mammary cells. Depletion of Rap1A expression significantly impaired LPA-stimulated migration of breast cancer cells and invasiveness in three-dimensional Matrigel cultures. Furthermore, we found that β-arrestin2 associates with the actin binding protein IQGAP1 in breast cancer cells, and is necessary for the recruitment of IQGAP1 to the leading edge of migratory cells. Depletion of IQGAP1 blocked LPA-stimulated breast cancer cell invasion. Finally, we have identified that LPA enhances the binding of endogenous Rap1A to β-arrestin2, and also stimulates Rap1A and IQGAP1 to associate with LPA₁. Thus our data establish novel roles for Rap1A and IQGAP1 as critical regulators of LPA-induced breast cancer cell migration and invasion.     

Tumor counterattack: fact or fiction?

Cancer development relies on a variety of mechanisms that facilitate tumor growth despite the presence of a functioning immune system. Understanding these mechanisms may foster novel therapeutic approaches for oncology and organ transplantation. By expression of the apoptosis-inducing protein CD95L (FasL, APO-1L, CD178), tumors may eliminate tumor-infiltrating lymphocytes and suppress anti-tumor immune responses, a phenomenon called tumor counterattack. On the one hand, preliminary evidence of tumor counterattack in human tumors exists, and CD95L expression can prevent T-cell responses in vitro. On the other hand, CD95L-expressing tumors are rapidly rejected and induce inflammation in mice. Here, we summarize and discuss the consequences of CD95L expression of tumor cells and its contribution to immune escape.This article is a symposium paper from the conference Tumor Escape and Its Determinants, held in Salzburg, Austria, on 10–13 October 2004.     

A Validated Mathematical Model of Tumor Growth Including Tumor–Host Interaction,Cell-Mediated Immune Response and Chemotherapy

We consider a dynamical model of cancer growth including three interacting cell populations of tumor cells, healthy host cells and immune effector cells. The tumor–immune and the tumor–host interactions are characterized to reproduce experimental results. A thorough dynamical analysis of the model is carried out, showing its capability to explain theoretical and empirical knowledge about tumor development. A chemotherapy treatment reproducing different experiments is also introduced. We believe that this simple model can serve as a foundation for the development of more complicated and specific cancer models.     

Endothelial-Rac1 Is Not Required for Tumor Angiogenesis unless αvβ3-Integrin Is Absent

Endothelial cell migration is an essential aspect of tumor angiogenesis. Rac1 activity is needed for cell migration in vitro implying a requirement for this molecule in angiogenesis in vivo. However, a precise role for Rac1 in tumor angiogenesis has never been addressed. Here we show that depletion of endothelial Rac1 expression in adult mice, unexpectedly, has no effect on tumor growth or tumor angiogenesis. In addition, repression of Rac1 expression does not inhibit VEGF-mediated angiogenesis in vivo or ex vivo, nor does it affect chemotactic migratory responses to VEGF in 3-dimensions. In contrast, the requirement for Rac1 in tumor growth and angiogenesis becomes important when endothelial β3-integrin levels are reduced or absent: the enhanced tumor growth, tumor angiogenesis and VEGF-mediated responses in β3-null mice are all Rac1-dependent. These data indicate that in the presence of αvβ3-integrin Rac1 is not required for tumor angiogenesis.