Lgr5-positive endothelial progenitor cells occupy a tumor and injury prone niche in the kidney vasa recta.

The Wnt pathway co-receptor, Leucine Rich Repeat Containing G Protein-Coupled Receptor 5 (LGR5), labels tumor-prone stem cell populations in certain types of tissue. In this study, we show that ARID1A and PIK3CA mutations in LGR5+ cells result in renal angiosarcomas in adult mice. The tumors originate in the renal medulla. We further show that LGR5 labels SOX17+/CD31+/CD34+/CD133+/AQP1+/CD146+ endothelial progenitor cells within the descending vasa recta or straight arterioles of the kidney, which are specialized capillaries that maintain medullary osmotic gradients necessary for water reabsorption and the production of concentrated urine. LGR5+ endothelial progenitor cells are tightly associated with contractile pericytes within the descending vasa recta. Long-term in vivo lineage tracing revealed that LGR5+ cells give rise to renal medullary vasculature. We further show that LGR5+ cells are activated in response to ischemic kidney injury. Our findings uncover a physiologically relevant endothelial progenitor cell population within the kidney vasa recta.

MET and ERBB2 are coexpressed in ERBB2+ breast cancer and contribute to innate resistance.

UNLABELLED: Breast cancer displays significant intratumoral heterogeneity, which has been shown to have a substantial impact on both innate and acquired resistance to tyrosine kinase inhibitors. The heterogeneous expression of multiple receptor tyrosine kinases (RTK) in cancers supports tumor signaling robustness and plays a significant role in resistance to targeted inhibition. Recent studies have revealed interactions between the MET receptor and the ERBB receptor family in the therapeutic resistance of several cancers. In this study, the relationship between MET expression/activity and the expression/activity of the ERBB receptor family in human breast cancer was interrogated. Importantly, a significant percentage of ERBB2(+) tumors coexpressing MET and ERBB2 were observed and displayed significant heterogeneity with subpopulations of cells that are MET(-)/ERBB2(+), MET(+)/ERBB2(-), and MET(+)/ERBB2(+). In a MET(+)/ERBB2(+) breast cancer cell line, MET depletion resulted in increased ERBB2 activation, and conversely, ERBB2 depletion resulted in increased MET activation. Neither EGFR nor ERBB3 compensated for MET or ERBB2 knockdown. The loss of either MET or ERBB2 led to a decrease in PI3K/AKT signaling and increased dependency on MAPK. These data show that a subset of ERBB2(+) breast cancers express MET and contain MET(+)/ERBB2(+) subpopulations. Moreover, analysis of RTK activation during ERBB2 knockdown indicated that MET signaling is a compensatory pathway of resistance. IMPLICATIONS: ERBB2(+) breast cancers with MET(+)/ERBB2(+) subpopulations may have an innate resistance to ERBB2 inhibition and may benefit from combined MET and ERBB2 inhibition.

Pericyte coverage of differentiated vessels inside tumor vasculature is an independent unfavorable prognostic factor for patients with clear cell renal cell carcinoma.

BACKGROUND: The objective of this study was to evaluate the effect of pericyte coverage (PC) of differentiated tumor microvessels on the prognosis of patients with clear cell renal cell carcinoma (CCRCC). METHODS: Samples from 2 cohorts of patients with CCRCC (101 Asian patients and 524 US patients) were prepared using 2 different histologic approaches: routine sectioning versus tissue microarray. Then, the samples were immunohistochemically doubled-stained for a pericyte marker (alpha smooth muscle actin [α-SMA]) and a differentiated vessel marker (cluster of differentiation 34 [CD34]), followed by multispectral image capturing and computerized image analyses to quantify the microvessel density (MVD) and the PC of differentiated vessels. The correlations of PC and the MVD:PC ratio with clinicopathologic characteristics were analyzed. RESULTS: There was an inverse correlation between differentiated MVD and PC. Higher PC correlated with more aggressive clinicopathologic characteristics of CCRCC in both cohorts, including more advanced T-classification, higher pathologic grades, and the occurrence of tumor necrosis. The MVD:PC ratio was an independent favorable prognostic factor for overall and recurrence-free survival in the Asian cohort and for recurrence-free survival in the US cohort. PC also was an independent prognostic factor, with higher PC predicting a poorer outcome. The combination of PC and MVD was better at distinguishing the outcome of patients with CCRCC. PC combined with differentiated MVD or with the MVD:PC ratio provided additional, independent prognostic information to the Leibovich risk model, and that information was used to generate improved risk models. CONCLUSIONS: The authors consistently observed that higher PC was correlated with more aggressive clinicopathologic characteristics. PC was an independent unfavorable prognostic factor. The authors concluded that pericytes should be considered for therapeutic targeting.

Molecular subtype analysis determines the association of advanced breast cancer in Egypt with favorable biology.

BACKGROUND: Prognostic markers and molecular breast cancer subtypes reflect underlying biological tumor behavior and are important for patient management. Compared to Western countries, women in North Africa are less likely to be prognosticated and treated based on well-characterized markers such as the estrogen receptor (ER), progesterone receptor (PR) and Her2. We conducted this study to determine the prevalence of breast cancer molecular subtypes in the North African country of Egypt as a measure of underlying biological characteristics driving tumor manifestations. METHODS: To determine molecular subtypes we characterized over 200 tumor specimens obtained from Egypt by performing ER, PR, Her2, CK5/6, EGFR and Ki67 immunohistochemistry. RESULTS: Our study demonstrated that the Luminal A subtype, associated with favorable prognosis, was found in nearly 45% of cases examined. However, the basal-like subtype, associated with poor prognosis, was found in 11% of cases. These findings are in sharp contrast to other parts of Africa in which the basal-like subtype is over-represented. CONCLUSIONS: Egyptians appear to have favorable underlying biology, albeit having advanced disease at diagnosis. These data suggest that Egyptians would largely profit from early detection of their disease. Intervention at the public health level, including education on the benefits of early detection is necessary and would likely have tremendous impact on breast cancer outcome in Egypt.

Development and evaluation of monoclonal antibodies against phosphatidylethanolamine binding protein 1 in pancreatic cancer patients.

Phosphatidylethanolamine binding protein 1 (PEBP1), also known as Raf kinase inhibitor protein (RKIP), has been considered as a suppressor of metastasis and a prognostic marker in prostate cancer, breast cancer, gastrointestinal stromal tumors, melanoma, and epithelial ovarian cancer. In this report, recombinant PEBP1 was successfully expressed in an Escherichia coli system. A panel of monoclonal antibodies (mAbs) against PEBP1 with high specificity and affinity was generated and characterized using ELISA, western blot analysis, immunofluorescent staining and immunohistochemical staining. PEBP1 expression in normal 293 cells and a few pancreatic cancer cell lines was detected with mAb 7F12 in western blot analysis. To screen for a pair of mAbs with optimal binding affinity to soluble PEBP1, ForteBio's Octet system was used. Sandwich ELISA with mAb pair 4F10 and 8E2 showed a linear correlation between absorbance and PEBP1 protein concentration over a range of 7 to 100 ng/ml. MAb 4A11 detected a high level expression of PEBP1 in normal pancreatic tissue, and cancer adjacent normal pancreatic tissue in a pancreatic tissue microarray (TMA) comprising 80 human tissue cores. Pancreatic cancer tissues show a no or very weak staining intensity of PEBP1. In 69 valid cases, PEBP1 expression was significantly lower in tumor than in normal pancreas (p=8.40E-14) and adjacent normal tissue (p=8.46E-17). PEBP1 expression in pancreatic cancer was not associated with pTMN stage, differentiation grade and pathologic diagnosis. In conclusion, our results suggest that PEBP1 overexpresses in normal pancreas but significantly decreases its expression in pancreatic cancer tissues. Anti-PEBP1 mAbs 4A11, 4F10, 7F12, and 8E2 are potential clinical diagnostic agents for pancreatic cancer.

A novel multipurpose monoclonal antibody for evaluating human c-Met expression in preclinical and clinical settings.

The inappropriate expression of the c-MET cell surface receptor in many human solid tumors necessitates the development of companion diagnostics to identify those patients who could benefit from c-MET targeted therapies. Tumor tissues are formalin fixed and paraffin embedded (FFPE) for histopathologic evaluation, making the development of an antibody against c-MET that accurately and reproducibly detects the protein in FFPE samples an urgent need. We have developed a monoclonal antibody (mAb), designated MET4, from a panel of MET-avid mAbs, based on its specific staining pattern in FFPE preparations. The accuracy of MET4 immunohistochemistry (MET4-IHC) was assessed by comparing MET4-IHC in FFPE cell pellets with immunoblotting analysis. The technical reproducibility of MET4-IHC possessed a percentage coefficient of variability of 6.25% in intra-assay and interassay testing. Comparison with other commercial c-MET antibody detection reagents demonstrated equal specificity and increased sensitivity for c-MET detection in prostate tissues. In cohorts of ovarian cancers and gliomas, MET4 reacted with ovarian cancers of all histologic subtypes (strong staining in 25%) and with 63% of gliomas. In addition, MET4 bound c-MET on the surfaces of cultured human cancer cells and tumor xenografts. In summary, the MET4 mAb accurately and reproducibly measures c-MET expression by IHC in FFPE tissues and can be used for molecular imaging in vivo. These properties encourage further development of MET4 as a multipurpose molecular diagnostics reagent to help to guide appropriate selection of patients being considered for treatment with c-MET-antagonistic drugs.

A highly invasive human glioblastoma pre-clinical model for testing therapeutics.

Animal models greatly facilitate understanding of cancer and importantly, serve pre-clinically for evaluating potential anti-cancer therapies. We developed an invasive orthotopic human glioblastoma multiforme (GBM) mouse model that enables real-time tumor ultrasound imaging and pre-clinical evaluation of anti-neoplastic drugs such as 17-(allylamino)-17-demethoxy geldanamycin (17AAG). Clinically, GBM metastasis rarely happen, but unexpectedly most human GBM tumor cell lines intrinsically possess metastatic potential. We used an experimental lung metastasis assay (ELM) to enrich for metastatic cells and three of four commonly used GBM lines were highly metastatic after repeated ELM selection (M2). These GBM-M2 lines grew more aggressively orthotopically and all showed dramatic multifold increases in IL6, IL8, MCP-1 and GM-CSF expression, cytokines and factors that are associated with GBM and poor prognosis. DBM2 cells, which were derived from the DBTRG-05MG cell line were used to test the efficacy of 17AAG for treatment of intracranial tumors. The DMB2 orthotopic xenografts form highly invasive tumors with areas of central necrosis, vascular hyperplasia and intracranial dissemination. In addition, the orthotopic tumors caused osteolysis and the skull opening correlated to the tumor size, permitting the use of real-time ultrasound imaging to evaluate antitumor drug activity. We show that 17AAG significantly inhibits DBM2 tumor growth with significant drug responses in subcutaneous, lung and orthotopic tumor locations. This model has multiple unique features for investigating the pathobiology of intracranial tumor growth and for monitoring systemic and intracranial responses to antitumor agents.

Mitogen-activated protein kinase kinase signaling promotes growth and vascularization of fibrosarcoma.

We hypothesized that signaling through multiple mitogen-activated protein kinase (MAPK) kinase (MKK) pathways is essential for the growth and vascularization of soft-tissue sarcomas, which are malignant tumors derived from mesenchymal tissues. We tested this using HT-1080, NCI, and Shac fibrosarcoma-derived cell lines and anthrax lethal toxin (LeTx), a bacterial toxin that inactivates MKKs. Western blots confirmed that LeTx treatment reduced the levels of phosphorylated extracellular signal-regulated kinase and p38 MAPK in vitro. Although short treatments with LeTx only modestly affected cell proliferation, sustained treatment markedly reduced cell numbers. LeTx also substantially inhibited the extracellular release of angioproliferative factors including vascular endothelial growth factor, interleukin-8, and basic fibroblast growth factor. Similar results were obtained with cell lines derived from malignant fibrous histiocytomas, leiomyosarcomas, and liposarcomas. In vivo, LeTx decreased MAPK activity and blocked fibrosarcoma growth. Growth inhibition correlated with decreased cellular proliferation and extensive necrosis, and it was accompanied by a decrease in tumor mean vessel density as well as a reduction in serum expression of angioproliferative cytokines. Vital imaging using high-resolution ultrasound enhanced with contrast microbubbles revealed that the effects of LeTx on tumor perfusion were remarkably rapid (<24 h) and resulted in a marked reduction of perfusion within the tumor but not in nontumor tissues. These results are consistent with our initial hypothesis and lead us to propose that MKK inhibition by LeTx is a broadly effective strategy for targeting neovascularization in fibrosarcomas and other similar proliferative lesions.

Anthrax lethal toxin inhibits growth of and vascular endothelial growth factor release from endothelial cells expressing the human herpes virus 8 viral G protein coupled receptor.

PURPOSE: In this study, we tested the hypothesis that inhibition of mitogen-activated protein kinase kinases (MKK) inhibits tumor growth by acting on angiogenic signaling and by extension may form the basis of an effective strategy for treatment of Kaposi's sarcoma. EXPERIMENTAL DESIGN: Murine endothelial cells expressing the human herpes virus 8 G protein-coupled receptor (vGPCR-SVEC) were treated with anthrax lethal toxin (LeTx). LeTx is a binary toxin ordinarily secreted by Bacillus anthracis and is composed of two proteins: protective antigen (the binding moiety) and lethal factor (the active moiety). Lethal factor is a protease that cleaves and inactivates MKKs. RESULTS: In vitro, treatment of vGPCR-SVEC with LeTx inhibited MKK signaling, moderately inhibited cell proliferation, and blocked the ability of these cells to form colonies in soft agar. Treatment with LeTx also blocked the ability of these cells to release several angioproliferative cytokines, notably vascular endothelial growth factor (VEGF). In contrast, inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase 1/2 with U0126 caused a substantial inhibition of proliferation but only modestly inhibited VEGF release. In xenograft models, i.v. injection of LeTx caused reduced tumor growth characterized immunohistochemically by inhibition of MKK signaling, decreased rates of proliferation, and reduced levels of VEGF and VEGF receptor 2, with a corresponding decrease in vascular density. CONCLUSIONS: These data support a role for MKK signaling in tumor growth and vascularization and are consistent with the hypothesis that inhibition of MKK signaling by LeTx or a similar agent may be an effective strategy for the treatment of Kaposi's sarcoma as well as other vascular tumors.

Inactivation of Apc in the mouse prostate causes prostate carcinoma.

Alterations of the Wnt/beta-catenin signaling pathway are positively associated with the development and progression of human cancer, including carcinoma of the prostate. To determine the role of activated Wnt/beta-catenin signaling in mouse prostate carcinogenesis, we created a mouse prostate tumor model using probasin-Cre-mediated deletion of Apc. Prostate tumors induced by the deletion of Apc have elevated levels of beta-catenin protein and are highly proliferative. Tumor formation is fully penetrant and follows a consistent pattern of progression. Hyperplasia is observed as early as 4.5 weeks of age, and adenocarcinoma is observed by 7 months. Continued tumor growth usually necessitated sacrifice between 12 and 15 months of age. Despite the high proliferation rate, we have not observed metastasis of these tumors to the lymph nodes or other organs. Surgical castration of 6-week-old mice inhibited tumor formation, and castration of mice with more advanced tumors resulted in the partial regression of specific prostate glands. However, significant areas of carcinoma remained 2 months postcastration, suggesting that tumors induced by Apc loss of function are capable of growth under conditions of androgen depletion. We conclude that the prostate-specific deletion of Apc and the increased expression of beta-catenin associated with prostate carcinoma suggests a role for beta-catenin in prostate cancer and offers an appropriate animal model to investigate the interaction of Wnt signaling with other genetic and epigenetic signals in prostate carcinogenesis.

Systemic anthrax lethal toxin therapy produces regressions of subcutaneous human melanoma tumors in athymic nude mice.

PURPOSE: Anthrax Lethal Toxin (LeTx), composed of protective antigen and lethal factor, catalytically cleaves mitogen-activated protein kinase (MAPK) kinases and inhibits the MAPK signaling pathways. The majority of metastatic melanomas possess the V599E BRAF mutation, which constitutively activates MAPK1/2 signaling. LeTx is cytotoxic to BRAF mutant melanoma cell lines in vitro, whereas most normal cells are resistant to this toxin. In this study, we determine the in vivo potency and safety of systemically administered LeTx. EXPERIMENTAL DESIGN: A s.c. xenograft melanoma model in athymic nude mice was treated with different i.p. doses of LeTx. RESULTS: In this study, we show that in vivo systemic LeTx treatment of s.c. xenograft melanoma tumors in athymic nude mice yields partial and complete tumor regressions with minor toxicity to mice. When animal toxicity was observed, we did not find any histologic evidence of tissue damage. CONCLUSIONS: LeTx is one of the rare targeted agents to produce complete remissions of human melanomas in an animal model and thus warrants further preclinical development.

Preparing the "soil": the primary tumor induces vasculature reorganization in the sentinel lymph node before the arrival of metastatic cancer cells.

Sentinel lymph node (SLN) metastasis is the first step in the spreading of cancer in many malignancies. Tumor-reactive lymphadenopathy in SLNs has been observed for decades, but alterations of the lymphatic channels and vasculature in these nodes before the arrival of metastatic tumor cells remain unexplored. Using animal models, we show here that, before the establishment of metastasis in the SLN, there are reorganizations of the lymphatic channels and the vasculature. The node becomes a functional blood vessel-enriched and lymph vessel/sinus-enriched organ before metastasis. The enlargement of the lymph sinuses is correlated with the primary tumor weight. The newly emerged functional blood vessels develop from high endothelial venules (HEV), in which the proliferation rate of the endothelial cells is also significantly increased. Similar alterations of the HEVs are also characterized in the axillary lymph nodes from human breast cancer patients without the evidence of metastasis. These findings support the hypothesis that modification of the microenvironment for a secondary tumor (i.e., vasculature reorganization in the SLN) can be initiated by a primary tumor before and independent of the physical presence of metastatic cancer cells.

Decreased BMD and limb deformities in mice carrying mutations in both Lrp5 and Lrp6.

UNLABELLED: Humans and mice lacking Lrp5 have low BMD. To evaluate whether Lrp5 and Lrp6 interact genetically to control bone or skeletal development, we created mice carrying mutations in both Lrp5 and the related gene Lrp6. We found that compound mutants had dose-dependent deficits in BMD and limb formation, suggesting functional redundancy between these two genes in bone and limb development. INTRODUCTION: Lrp5 and Lrp6 are closely related members of the low density lipoprotein receptor family and are co-receptors for Wnt ligands. While Lrp5 mutations are associated with low BMD in humans and mice, the role of Lrp6 in bone formation has not been analyzed. MATERIALS AND METHODS: To address whether Lrp5 and Lrp6 play complimentary roles in bone and skeletal development, we created mice with mutations in both genes. We inspected limbs of mice from the different genotypic classes of compound mutants to identify abnormalities. DXA and muCT were used to evaluate the effect of mutations in Lrp5 and Lrp6 on BMD and microarchitecture. RESULTS: Mice heterozygous for mutations in Lrp6 and either heterozygous or homozygous for a mutation in Lrp5 (Lrp6(+/-);Lrp5(+/-) or Lrp6(+/-);Lrp5(-/-)) display limb defects with incomplete penetrance and variable expression. DXA analysis showed that BMD decreased as mice progressively were more deficient in Lrp5 and Lrp6. Lrp6(+/-);Lrp5(-/-) mice were more severely affected than Lrp6(+/+);Lrp5(-/-) mice, whereas Lrp6(+/-);Lrp5(+/-) mice had statistically higher BMD than Lrp6(+/+);Lrp5(-/-) mice and lower BMD compared with wildtype mice and mice heterozygous for either mutation alone. CONCLUSIONS: Lrp6 and Lrp5 genetically interact in limb development in mice. Furthermore, heterozygosity for an inactivating mutation in Lrp6 further reduces BMD in both male and female mice lacking Lrp5.