Development Of Metastatic Tumors Research Articles

Cyclophosphamide Creates a Receptive Microenvironment for Prostate Cancer Skeletal Metastasis

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

Abstract 3295: Systematic in vivo analysis of PI3K pathway aberrations in a mouse model for invasive lobular carcinoma

Abstract Invasive lobular carcinoma (ILC) is a form of breast cancer that develops in the milk-producing glands (lobules) of the breast. ILC is an aggressive form of breast cancer due to its ability to spread to other parts of the body, both by bloodstream as well as lymphatic system. One of the key features found in ILC is loss of E-cadherin, encoding CDH1. Mammary-gland specific deletion of E-cadherin does not lead to tumour formation indicating that other factors are involved in the induction of ILC. Indeed, combined tissue-specific inactivation of E-cadherin and p53 induces development of invasive and metastatic mammary tumours resembling human ILC. However, a limitation of the current conditional mouse models is that human ILCs rarely contain p53 mutations. In contrast, PI3K pathway mutations are frequently found in human ILCs. Currently we are developing novel mouse ILC models to systematically examine the contribution of PI3K pathway components such as PIK3CA, AKT and PTEN to ILC development. To reduce time spent on generating a genetically engineered mouse (GEM) tumour cohort expressing three or more transgenes we set-up embryonic stem (ES) cell derivation and targeting of ES cells from validated GEM models, the so-called GEMM-ESC strategy. WapCre-Cdh1F/F ES cells targeted with PIK3CA or AKT mutants are now being used to generate the elaborate PI3K pathway tumour cohort. These advanced models will allow us to study the role of the PI3K pathway in the pathogenesis of ILC. Moreover, we can use these advanced mouse models which recapitulate key hallmarks of human ILC to perform refined and realistic preclinical prevention and intervention studies for ILC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3295. doi:1538-7445.AM2012-3295

Chronic occupational exposure to arsenic induces carcinogenic gene signaling networks and neoplastic transformation in human lung epithelial cells

Chronic arsenic exposure remains a human health risk; however a clear mode of action to understand gene signaling-driven arsenic carcinogenesis is currently lacking. This study chronically exposed human lung epithelial BEAS-2B cells to low-dose arsenic trioxide to elucidate cancer promoting gene signaling networks associated with arsenic-transformed (B-As) cells. Following a 6month exposure, exposed cells were assessed for enhanced cell proliferation, colony formation, invasion ability and in vivo tumor formation compared to control cell lines. Collected mRNA was subjected to whole genome expression microarray profiling followed by in silico Ingenuity Pathway Analysis (IPA) to identify lung carcinogenesis modes of action. B-As cells displayed significant increases in proliferation, colony formation and invasion ability compared to BEAS-2B cells. B-As injections into nude mice resulted in development of primary and secondary metastatic tumors. Arsenic exposure resulted in widespread up-regulation of genes associated with mitochondrial metabolism and increased reactive oxygen species protection suggesting mitochondrial dysfunction. Carcinogenic initiation via reactive oxygen species and epigenetic mechanisms was further supported by altered DNA repair, histone, and ROS-sensitive signaling. NF-κB, MAPK and NCOR1 signaling disrupted PPARα/δ-mediated lipid homeostasis. A ‘pro-cancer’ gene signaling network identified increased survival, proliferation, inflammation, metabolism, anti-apoptosis and mobility signaling. IPA-ranked signaling networks identified altered p21, EF1α, Akt, MAPK, and NF-κB signaling networks promoting genetic disorder, altered cell cycle, cancer and changes in nucleic acid and energy metabolism. In conclusion, transformed B-As cells with their whole genome expression profile provide an in vitro arsenic model for future lung cancer signaling research and data for chronic arsenic exposure risk assessment.

Akt/PKB-Mediated Phosphorylation of Twist1 Promotes Tumor Metastasis via Mediating Cross-Talk between PI3K/Akt and TGF-β Signaling Axes

Metastatic breast tumor cells display an epithelial-mesenchymal transition (EMT) that increases cell motility, invasion, and dissemination. Although the transcription factor Twist1 has been shown to contribute to EMT and cancer metastasis, the signaling pathways regulating Twist1 activity are poorly understood. Here, we show that Twist1 is ubiquitously phosphorylated in 90% of 1,532 invasive human breast tumors. Akt/protein kinase B (PKB)-mediated Twist1 phosphorylation promotes EMT and breast cancer metastasis by modulating its transcriptional target TGF-β2, leading to enhanced TGF-β receptor signaling, which in turn maintains hyperactive phosphoinositide 3-kinase (PI3K)/Akt signaling. Preventing phosphorylation of Twist1, as well as depletion of TGF-β2, significantly impaired the metastatic potential of cancer cells in vivo, indicating a key role of phosphorylated Twist1 (phospho-Twist1) in mediating cross-talk between the PI3K/Akt and TGF-β/Smad signaling axes that supports metastatic tumor development. Our results describe a novel signaling event linking PI3K/Akt hyperactivation in tumor cells to direct regulation of Twist1 activation and tumor metastasis. We identified the first phospho-Twist1 transcriptional target TGF-β2, which mediates cross-talk between PI3K/Akt and TGF-β signaling and promotes tumor metastasis. Our results thus illustrate a direct role of PI3K/Akt signaling in metastatic cancer development and suggest that Twist1 phosphorylation could be a potential therapeutic target in clinical cancer treatment.

Uveal Melanoma—Clinical and Pathological Update

Although ocular treatments of the primary uveal melanoma are effective to eradicate the primary tumor locally, its impact on survival remains unclear. The reason we believe local treatment has limited or no impact on survival is because, despite the increase in diagnostic accuracy over the past several decades and development of globe-sparing treatments, the mortality rates of patients with uveal melanomas of any given size have not changed appreciably during this period. Development of metastases is a complex multi-step process that occurs almost exclusively via a hematogenous route. Due to the complexity of this process it is difficult to isolate individual events in patients that are important in the overall progression of the disease from primary tumor to metastasis. Because most uveal melanomas are currently managed by eye-preserving methods that do not yield a pathologic specimen, a number of investigators are currently performing biopsy (usually fine needle aspiration biopsy) of these tumors prior to, or at the time of, ocular treatment to obtain representative tumor specimens for cytopathologic, cytogenetic, and/or molecular biologic analysis. Many new discoveries about the cytogenetic and molecular biologic abnormalities in uveal melanoma cells, and the ways these features influence development and prognosis of metastatic tumors have been published. Hopefully these findings will help us understand the process and improve patient survival since the impact of surveillance testing for metastasis and systemic therapy on the survival of patients with metastatic uveal melanoma is still uncertain.

Role of parathymic lymph nodes in metastatic tumor development

Parathymic lymph nodes as potential sites of tumor progression have been neglected in humans. We have established a rat renal capsule-parathymic lymph node model to study in vivo metastasis. Epithelial liver carcinoma (HeDe) and mesenchymal mesoblastic nephroma (NeDe) cell lines have been established after inducing chemical carcinogenesis in newborn Fisher 344 inbred rats by N-nitrosodimethylamine. Implanting the exact number of tumor cells (HeDe, NeDe) under the renal capsule allowed the standardization and timing of metastatic development. Tumor cells released from the primary tumor in the peritoneal cavity were drained to the parathymic lymph nodes (PTNs) as sentinel lymph nodes. Similarly, tumor cells injected i.p. were engulfed by macrophages, drained through the transdiaphragmatic channels, and transported to the thoracal lymphatics, primarily to PTNs. Tumor cells after transdiaphragmic drainage can enter both anterior mammary and parathymic sentinel lymph nodes. The potential common origin can shed new light on the metastatic cell progression of PTNs and mammary tumors.

Strategy of radiation therapy for bone metastases and MSCC in breast cancer patients

Bone metastasis is a common event in advanced cancers such as prostate, breast, lung, and renal cancers. Radiation therapy has been widely used for bone metastasis. However, it remains a challenging therapy because no radiation therapeutic guidelines, including radiation dose, radiation field, and fractionation, for patients with bone metastasis have been established. Many randomized controlled trials for bone metastasis have been carried out. They showed no significant difference in pain relief with a short course of radiation therapy such as 8Gy/1Fr and 20Gy/5Fr or with a long course of radiation therapy such as 30Gy/10Fr, 37.5Gy/15Fr, and 40Gy/20Fr. Toxicity rates with short and long courses were also the same. Recurrence rate at 2years, however, was significantly higher in patients irradiated with a short course than in patients irradiated with a long course. Those trials also showed that response rate is affected by patient's age, performance state, tumor type, pathological state, number of metastatic tumors, and span from diagnosis of cancer to development of metastatic tumor. Breast cancer has a better prognosis than most other cancers. Recently, there have been significant advances in cancer therapy techniques and improvement in clinical results. Bone metastasis can cause extreme pain and motor deficits. Quality of life for patients with bone metastasis is drastically worsened. Patients with bad prognosis should be treated with radiation therapy when analgesia is the main aim of treatment. Survival of patients with oligometastasis or predominantly bone metastasis is expected to be better than that of patients with visceral metastasis. For patients with vertebral or weight-bearing long bone metastasis, long-course therapy is recommended. Many patients who are expected to have a good prognosis should be treated with a long course of radiation.

Telomere Dysfuction Promotes Metastasis in a TERC Null Mouse Model of Head and Neck Cancer

Squamous cell carcinoma arises from highly proliferative basal layer epithelial cells, which normally divide for a short time before detaching from the basement membrane and undergoing terminal differentiation. Basal layer cells in stratified epithelia express the reverse transcriptase known as telomerase. Most human cells do not express telomerase and therefore are subject to loss of telomeric DNA with age due to the inability of lagging strand synthesis to completely replicate chromosomal ends. Late generation telomerase deficient mice exhibit signs of premature aging including reduced function of proliferating cellular compartments. We examined development of squamous cell carcinoma in a telomerase deficient murine background with long and short telomeres. G1 Terc-/- mice (long telomeres) had fewer lymph node metastases, which correlated with increased numbers of apoptotic cells in these tumors compared with wild-type mice. However, G5 Terc-/- mice with short telomeres had increased metastatic tumor burden similar to wild type mice. This increased metastasis correlated with genomic instability and aneuploidy in tumor cells from G5 Terc-/- mice. A number of similarities with human SCC were noted in the mouse model, and dramatic differences in global gene expression profiles were shown between primary and metastatic tumors. We concluded that telomere shortening promotes metastatic tumor development in a Terc null mouse model of head and neck cancer.

Abstract 1589: Development of a mouse model for bone marrow disseminated tumor cells with human tumor xenografts

Abstract Objective: Disseminated tumor cells (DTCs) found in the bone marrow (BM) of breast cancer patients portends a poor prognosis. BM DTCs are thought to be intermediaries in the metastatic process. Study of these cells has been limited due to their scarcity. To overcome this limitation, we have developed an animal model for DTCs employing the Human and Mouse Linked Evaluation of Tumor (HAMLET) system. Experimental procedures: After informed consent, human breast adenocarcinomas were prospectively collected from 5 patients with estrogen receptor negative/Her2 negative tumors and implanted into a humanized NOD/SCID mouse mammary fat pad as previous described. BM was collected from the femur and tibia from mice at varying passages of the tumors and analyzed for human-specific GADPH (hGAPDH) expression by qRT-PCR. The presence of other human gene transcripts, previously detected in the BM of breast cancer patients and believed to be associated with the presence of DTCs, was also determined by qRT-PCR. Human gene expression array analysis (Affymetrix Human gene 1.0ST) was performed on the primary xenograft tumor, a spleen metastatic nodule and BM from all WHIM17 mice. Results: BM was screened from 16 tumor bearing mice for the presence of DTCs. 10 animals, with tumors derived from 2 patients, developed metastatic disease and 9 had detectable levels of hGAPDH. The CT ratio human to mouse GAPDH varied from 1.06 to 2.23. In particular, high levels of hGAPDH were found in one HAMLET mouse line, WHIM 17, in which all animals developed metastases (CT ratio human to mouse GAPDH=1.06 to 1.31). One mouse with detectable hGAPDH died of other causes and could not be assed for metastatic disease development. There was no detectable hGAPDH in five mice, derived from 3 patient tumors, which did not develop metastatic tumors and in control mice with humanized mammary fat pads. Human-specific transcripts for SNAIL1, GSC, FOXC2 and KRT19 were detectable in the BM of the WHIM 17 mice whereas control, non-tumor bearing humanized mice demonstrated no expression of any of these markers. Microarray analysis confirmed the presence of human cells in mouse BM, allowing the identification of genes specifically associated with BM DTC cell populations. Conclusion: We have utilized a mouse xenograft model for the study of DTCs using primary human breast adenocarcinomas transplanted into NOD/SCID mice. The presence of human cells in mice BM appears to correlate with metastatic tumor development. The gene expression patterns of these cells indicate that they maintain molecular profiles similar to DTCs isolated directly from the BM of breast cancer patients. We believe that this model will allow for a better understanding of the metastatic process in breast cancer patients and provide an in vivo model for monitoring and assessing the efficacy of new therapeutic agents in eradicating micrometastatic disease and ultimately, improving survival in breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1589. doi:10.1158/1538-7445.AM2011-1589

Dynamic molecular changes associated with epithelial–mesenchymal transition and subsequent mesenchymal–epithelial transition in the early phase of metastatic tumor formation

Metastatic tumor formation via vessel route begins with cancer cell extravasation from vessel lumen, migration into the connective tissue surrounding vessels, and invasion into target organ parenchyma. Epithelial-mesenchymal transition (EMT) and mesenchymal-epithelial transition (MET) have been recognized to play an important role in metastatic process, however, how and where these biological changes take place in the early phase of metastatic tumor development has never been clarified. We morphologically evaluated 34 small intrapulmonary metastases formed after cancer cell extravasation from lymphatics (lymphogenic metastasis) and 40 formed in the absence of extravasation (aerogenous metastasis) in human specimens and found that isolated or small clusters of invasive cancer cells (tumor budding) were frequently observed in lymphogenic metastasis (24/34; 71%), but were never observed within aerogenous metastasis. We immunostained 34 lymphogenic metastases for 13 molecular markers of EMT and MET and scored the immunostaining intensity of cancer cells floating in lymphatic vessels (LVs), migrating into the connective tissue surrounding vessels [bronchovascular bundle (BVB)], and growing in lung parenchyma (LP). Cancer cells within BVBs stained more weakly for E-cadherin (p < 0.001), β-catenin (p < 0.001), and Geminin (p < 0.001) and more strongly for MMP-7 (p = 0.046) and Laminin-5 γ2 (p = 0.037) than tumor cells in LVs. However, cancer cells in LP exhibited resurgent E-cadherin (p = 0.011), β-catenin (p < 0.001), and Geminin (p = 0.037) expression and reduced MMP-7 (p = 0.038) and Laminin-5 γ2 (p = 0.001) expression in comparison with cancer cells in BVBs. Our results suggested that in the early phase of metastatic tumor formation cancer cells undergo dynamic phenotypic change associated with EMT and subsequent MET.

CC3/TIP30 regulates metabolic adaptation of tumor cells to glucose limitation

CC3/TIP30 is a metastasis and tumor suppressor, with reduced or absent expression in a variety of aggressive tumors. Overexpression of CC3 in tumor cells predisposes them to apoptosis in response to different death signals. We found that silencing of CC3 expression does not increase apoptotic resistance of cells. However, it strongly improves survival of tumor cells in response to glucose limitation. HeLa cells with silenced CC3 survive long-term in low glucose, and, in comparison to control HeLa cells, show superior metabolic adaptation to glucose limitation. First, unlike the parental HeLa cells, HeLa with silenced CC3 activate and maintain high levels of mitochondrial respiration that is critical for their ability to thrive in low glucose. Second, silencing of CC3 leads to higher expression levels of mitochondrial proteins in respiration complexes when cells are continuously cultured in limiting glucose. Third, HeLa cells with silenced CC3 maintain higher levels of c-MYC and the M2 isoform of pyruvate kinase in low glucose, contributing to more efficient glycolysis. Fourth, HeLa cells with silenced CC3 fail to fully activate AMPK in response to glucose limitation. Inhibition of AMPK, either pharmacologic or via siRNA, protects control HeLa cells from death in low glucose. The metabolic flexibility acquired by cells after silencing of CC3 could be directly relevant to the development of metastatic and aggressive human tumors that frequently have low or absent expression of CC3.

Abstract LB-325: Antitumor effect of resveratrol combined with immunocytokine immunotherapy

Abstract Resveratrol (RV) is a natural compound which is currently being investigated for many possible health promoting activities. It has anti-tumor activity on cancer cells in vitro and in vivo in some animal models of cancer. We are currently examining the ability of RV to be combined with immunotherapy to treat cancer. Our immunotherapy is the hu14.18-IL2 immunocytokine (IC), consisting of the hu14.18 mAb (that targets GD2 on the surface of some tumor cells) linked to interleukin-2 (IL2). The IL2 component attracts and activates immune effector cells expressing IL2 receptors and induces localized destruction of the tumor via antibody dependent cell-mediated cytotoxicity (ADCC) by NK cells. Prior studies have found RV to be immunosuppressive in vitro but not in vivo. RV at a daily intragastric (i.g.) dose of 50mg/kg (1mg per mouse) slowed tumor growth in mice bearing neuroblastoma and was not immunosuppressive. At this dose the peak serum RV level was ∼ 1mcM. Using proliferative and cytotoxicity assays we were defining a dose in vitro and a regimen in vivo that does not suppress the immune system and is additive or synergistic in mediating anti-tumor effects when combined with immunotherapy. In our in vitro studies high concentrations of RV (≥ 25mcM) were found to inhibit both tumor cell proliferation and the ability of PHA and IL2 to stimulate proliferation of human and murine effector cells (measured by 3H-thymidine incorporation and by dilution of CFSE in daughter cells). These cells were arrested in their G1 phase. In addition, these high RV concentrations prevented effector cells from mediating ADCC in vitro. In contrast, 1mcM (as found in serum after i.g. treatment) was not tumoristatic or immunosuppressive in vitro. Systemic RV regimens, although inhibiting tumor growth in vivo, did not induce tumor regression in these models, probably because of the limited amount of RV reaching the tumor. In order to increase the amount of RV in the tumor, studies on the anti-tumor effect of peritumoral (p.t.) injections of 20mg RV in vivo in NXS2 neuroblastoma-bearing mice were performed. The results of these studies showed that peritumoral RV had a robust but transient anti-tumor effect. Primary subcutaneous tumors initially regressed; however, most mice then developed metastatic tumors, and in some cases re-growth of the primary tumor, after cessation of RV therapy. In order to circumvent tumor recurrence and metastasis, intravenous (i.v) immunotherapy was combined with the p.t. injections of 20mg RV. Peritumoral injection with resveratrol enhanced the subsequent efficacy of immunotherapy. Further, most mice receiving p.t. RV in combination with IC not only resolved their primary tumors, but also survived without development of metastatic tumors. Sixty-one % of the mice receiving the combined therapy showed no detectable tumors by day 100 compared to 15-13% of the mice in the RV-alone or the IC-alone groups, respectively (p=0.001 and p=0.0003). These studies indicate the potential for a combined regimen of RV and hu14.18-IL2. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-325.

Abstract 5120: A6 peptide binds to CD44 and inhibits migration and metastasis of CD44+ cell lines in in vitro and in vivo studies

Abstract Background: CD44 plays a role in many physiological and pathological processes, including cell-cell and cell-extracellular matrix interactions, cell migration, and metastatic spread. A6 (KPSSPPEE) is a synthetic peptide that inhibits migration of human cancer cell lines and has anti-tumor activity in the absence of significant adverse events in tumor models and in a Phase 2 clinical trial in ovarian cancer. The current study was directed at identifying the mechanism of action of A6 through investigation of its effects on cell migration and metastasis. Methods: The effect of A6 on migration was investigated using human ovarian and breast carcinoma cell lines. Effects on cell migration in response to NIH3T3 conditioned media supplemented with VEGF were assessed using the Boyden chamber technique. To demonstrate the potential of A6 to inhibit cancer metastasis in vivo, the number of tumor nodules generated in the lungs of C57BL/6 mice intravenously inoculated with B16-F10 mouse melanoma cells was quantified. To characterize A6 binding and CD44 expression, standard FACS, cross-linking, and immunoprecipitation/blotting techniques were used. Results: A6 inhibited the migration of a subset of ovarian and breast cancer cell lines (e.g. SKOV3, OVCAR3, MDA-MB 361 and MDA-MB 468), exhibiting IC50 values in an in vitro chemotaxis assay in the pM to nM range. The ability of A6 to inhibit cancer cell migration in vitro correlated with CD44 expression: CD44 positive lines (e.g., SKOV3, OVCAR3, murine B16-F10 melanoma) were inhibited by A6 whereas cell lines expressing little or no CD44 (e.g., OVCAR4, OVCAR5, late passage A2780) were refractory to A6. Immunopreciptation studies demonstrate that CD44 binds biotin-tagged A6 peptide cross-linked to the plasma membrane. Although A6 perturbed the binding of a monoclonal anti-CD44 antibody (DF-1485) to SKOV3 cells it did not inhibit hyaluronic acid (HA)-induced adhesion of cancer cells through CD44 suggesting that A6 peptide is not a competitive antagonist of CD44/HA binding. In fact A6 potentiated the HA-induced adhesion of cancer cells. The ability of A6 to agonize the CD44 receptor and desensitize cells to migration signals represents a novel strategy for inhibiting CD44 function. In vivo, A6 (100 mg/kg delivered subcutaneously twice a day for 11 days) reduced the number of lung foci generated from the IV inoculation of 106 B16-F10 melanoma cells by 50% (pvalue= 0.003 in an unpaired t test), in a lung metastasis model dependent on the migration of B16-F10 melanoma cells from the circulation to the lungs. Conclusions: A6 potently blocks of the migration of CD44-positive cells in vitro through a novel mechanism of action. It binds to CD44 on the cell surface and in vivo it blocks the development of metastatic tumors at secondary sites. GOG study 170N is testing the activity of A6 in patients with recurrent ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5120.

Abstract LB-366: A role for hNDPK-B in breast cancer metastasis in vivo.

Abstract Cancer cells promote angiogenesis early in tumorigenesis under mutagenic/hypoxic conditions that drive oncogene expression of pro-angiogenic proteins. Clinical data suggest that by the time women are diagnosed with breast cancer, micrometastases are extant but suppressed by the primary tumor. It is imperative that we discover how to suppress metastases at or before breast biopsy, lumpectomy or mastectomy. We have shown in vitro that human breast cancers secrete nucleoside diphosphate kinase (sNDPK), which generates ATP from ADP locally to act at ATP-P2Y receptors locally to regulate blood flow, cell migration, and vascular permeability consistent with intravasation and extravasation of cancer cells and angiogenic stimulation at the metastatic niche. The ability of ATP-P2 receptor stimulation to transactivate the endothelial VEGF receptor in the absence of VEGF suggests the possibility that cancer cell-secreted NDPK, endothelial P2Y receptors, and VEGF receptors interact as partners in tumor angiogenesis in vitro. Our in vitro data strongly suggest a role for NDPK blockers and P2-receptor antagonists in adjuvant therapy for breast cancer (US Patent No. 10/331,375). We have now extended our studies of purinergic regulation of angiogenesis in vivo in a breast cancer metastasis model in mice carrying luciferase-tagged human breast carcinoma cells (MDA-MB-231Luc+) to permit tracking of primary tumor development and tumor metastases. We hypothesized that orthotopic-human tumors growing in SCID mice modulate extracellular sNDPK-B levels to attract angiogenic growth, development and metastasis. Examination of serum using Western blot and an ELISA assay of our own design revealed that tumors secreted sNDPK-B in large amounts two weeks after subcutaneous injection of cancer cells in the mammary fat pad. There is a correlation between secreted NDPK-B and tumor size. We confirmed the activity of sNDPK-B as a phosphoprotein trans-phosphorylase. PCR studies suggest that in more aggressive tumors, expression of sNDPK-B mRNA is increased (r2=0.97), while there is no correlation between intracellular NDPK-B and extracellular sNDPK-B expression. Differential mRNA expression may be seen between primary and metastatic cells and could underscore the role of sNDPK-B in mediating metastatic tumor development. Studies underway with mice carrying tumors and implanted with osmotic drug-delivery pumps will determine the effect of NDPK-B blockers, purinergic receptor antagonists and combined therapy on tumor burden and metastasis development and regression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-366.

Induction of metastatic gastric cancer by peroxisome proliferator-activated receptorδ activation.

Peroxisome proliferator-activated receptorδ (PPARδ) regulates a multiplicity of physiological processes associated with glucose and lipid metabolism, inflammation, and proliferation. One or more of these processes likely create risk factors associated with the ability of PPARδ agonists to promote tumorigenesis in some organs. In the present study, we describe a new gastric tumor mouse model that is dependent on the potent and highly selective PPARδ agonist GW501516 following carcinogen administration. The progression of gastric tumorigenesis was rapid as determined by magnetic resonance imaging and resulted in highly metastatic squamous cell carcinomas of the forestomach within two months. Tumorigenesis was associated with gene expression signatures indicative of cell adhesion, invasion, inflammation, and metabolism. Increased PPARδ expression in tumors correlated with increased PDK1, Akt, β-catenin, and S100A9 expression. The rapid development of metastatic gastric tumors in this model will be useful for evaluating preventive and therapeutic interventions in this disease.

A tissue reconstitution model to study cancer cell‐intrinsic and ‐extrinsic factors in mammary tumourigenesis

The contribution of cancer cell-intrinsic and -extrinsic factors to metastatic breast cancer is still poorly understood, hampering development of novel therapeutic strategies that decrease breast cancer mortality. Cre/loxP-based conditional mouse models of breast cancer present unique opportunities to study sporadic tumour formation and progression in a controlled setting. Unfortunately, the generation of mouse strains carrying multiple mutant alleles needed for such studies is very time-consuming. Moreover, conditional mouse tumour models do not permit independent manipulation of tumour cell-intrinsic and -extrinsic factors. Although the latter can be achieved by cleared fat-pad transplantation of mouse mammary epithelial cells (MMECs) from tumour suppressor gene (TSG) knockouts into wild-type or mutant recipients, this procedure is not possible for mutations that cause embryonic lethality or preclude mammary gland development. Here we show that cleared fat-pad transplantations with MMECs isolated from K14cre;Cdh1(F/F); Trp53(F/F) mice expressing Cre recombinase under control of the cytokeratin-14 promoter and carrying conditional null alleles for p53 and E-cadherin (Cdh1) first resulted in the formation of phenotypically normal mammary glands, followed by the development of invasive metastatic mammary tumours. Tumour formation in the recipients mimicked tumour latency, spectrum, morphology, immunophenotype, and metastatic characteristics of the original mammary tumour model. This transplantation system, which can be expanded to other conditional TSG knockouts, permits independent genetic analysis of stromal factors and testing of additional cancer cell-intrinsic mutations that would otherwise be embryonic lethal or require intensive breeding.

A Genetic Variant of Aurora Kinase A Promotes Genomic Instability Leading to Highly Malignant Skin Tumors

Aurora kinase A (Aurora-A) belongs to a highly conserved family of mitotis-regulating serine/threonine kinases implicated in epithelial cancers. Initially we examined Aurora-A expression levels at different stages of human skin cancer. Nuclear Aurora-A was detected in benign lesions and became more diffused but broadly expressed in well and poorly differentiated squamous cell carcinomas (SCC), indicating that Aurora-A deregulation may contribute to SCC development. To mimic the overexpression of Aurora-A observed in human skin cancers, we established a gene-switch mouse model in which the human variant of Aurora-A (Phe31Ile) was expressed in the epidermis upon topical application of the inducer RU486 (Aurora-AGS). Overexpression of Aurora-A alone or in combination with the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA), did not result in SCC formation in Aurora-AGS mice. Moreover, Aurora-A overexpression in naive keratinocytes resulted in spindle defects in vitro and marked cell death in vivo, suggesting that the failure of Aurora-A to initiate tumorigenesis was due to induction of catastrophic cell death. However, Aurora-A overexpression combined with exposure to TPA and the mutagen 7,12-dimethylbenz(a)anthracene accelerated SCC development with greater metastatic activity than control mice, indicating that Aurora-A cannot initiate skin carcinogenesis but rather promotes the malignant conversion of skin papillomas. Further characterization of SCCs revealed centrosome amplification and genomic alterations by array CGH analysis, indicating that Aurora-A overexpression induces a high level of genomic instability that favors the development of aggressive and metastatic tumors. Our findings strongly implicate Aurora-A overexpression in the malignant progression of skin tumors and suggest that Aurora-A may be an important therapeutic target.

Conditional deletion of Smad1 and Smad5 in somatic cells of male and female gonads leads to metastatic tumor development in mice.

The transforming growth factor beta (TGFbeta) family has critical roles in the regulation of fertility. In addition, the pathogenesis of some human cancers is attributed to misregulation of TGFbeta function and SMAD2 or SMAD4 mutations. There are limited mouse models for the BMP signaling SMADs (BR-SMADs) 1, 5, and 8 because of embryonic lethality and suspected genetic redundancy. Using tissue-specific ablation in mice, we deleted the BR-SMADs from somatic cells of ovaries and testes. Single conditional knockouts for Smad1 or Smad5 or mice homozygous null for Smad8 are viable and fertile. Female double Smad1 Smad5 and triple Smad1 Smad5 Smad8 conditional knockout mice become infertile and develop metastatic granulosa cell tumors. Male double Smad1 Smad5 conditional knockout mice are fertile but demonstrate metastatic testicular tumor development. Microarray analysis indicated significant alterations in expression of genes related to the TGFbeta pathway, as well as genes involved in infertility and extracellular matrix production. These data strongly implicate the BR-SMADs as part of a critical developmental pathway in ovaries and testis that, when disrupted, leads to malignant transformation.

Angiogenesis and breast cancer

The growth of breast cancers, as is the case in other solid tumors, is dependent on the development of neo-vessels. This has been clearly demonstrated in preclinical models and clinical-biological studies. Yet, the biological mechanisms involved in angiogenesis are heterogeneous; pathways activated by vascular endothelial growth factor (VEGF) seem to play a prominent role in the early stages of the disease, whereas a number of other pathways are involved in the development of progressive and metastatic tumors. This might account for the disappointing results of phase III studies of bevacizumab in breast cancer as compared with results reported in renal or colon cancers. The use of new drugs targeting other angiogenic factors and a better selection of patients eligible for treatment might improve these results. This is a review of the major biological and clinical data available on the critical role of angiogenesis in breast cancer. We will also review the different clinical studies reported to date in this area.

Genomic Instability and the Development of Metastatic Lymph Node Tumors

Although recent data suggest that cells with metastatic potential disseminate from the primary breast tumor early in tumor development, the mechanism by which disseminated breast cancer cells proliferate within foreign tissues is not well understood. Here, we examined levels and patterns of allelic imbalance (AI) in metastatic lymph node (LN) tumors to identify molecular signals that promote the survival and growth of disseminated breast tumor cells. DNA from 106 metastatic LN tumors from 25 patients was isolated after laser microdissection of pure tumor cell populations. AI was assessed at 26 chromosomal regions frequently altered in breast cancer. Tumor burden was calculated by dividing the area of the metastatic tumor in the node by the area of the entire LN. Metastatic tumor burden ranged from focal to complete replacement of the LN with tumor. Grouping the nodes as < 25% tumor, 25-50% tumor, 50-75% tumor, and > or = 75% tumor replacement revealed the average frequency of AI ranged from 0.13 (+/-0.11) in the < 25% group to 0.17 (+/-0.13) in LNs with > or = 75% tumor burden. The range of AI in both the < 25% and > 75% replacement group was 0.00-0.48. Allelic losses at chromosomal regions 1p36.1-36.2, 5q21.1-21.3, 6q15, 10q23.31-23.33, and 17p13.1 were significantly higher in metastatic LNs with > 75% compared with < 25% tumor burden. In metastatic LNs, levels of AI were not associated with tumor burden, suggesting that accumulation of genetic changes is not coincidental with tumor growth; rather the accumulation of specific genetic changes is a prerequisite to the transformation of disseminated breast cells into metastatic LN tumors.