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A375Br cells were transfected with one of four siRNA clones directed against PLEKHA5 or a nontargeting control siRNA. Knock-down is demonstrated by Western blot.

Purpose: Approximately 40% of patients with metastatic melanoma develop brain metastases. Our purpose was to identify genes aberrantly expressed in melanoma that might be associated with propensity for brain homing.Experimental Design: We studied gene expression profiles in a cell line model of brain metastasis (cerebrotropic A375Br cells vs. parental A375P cells) and compared them with profiles of patients who developed early brain metastases and who did not. A tissue microarray containing 169 metastatic melanoma cases with variable time to brain metastasis was constructed to further study marker expression by quantitative immunofluorescence. An in vitro model of the blood brain barrier (BBB) was generated to evaluate potential mediators of brain metastases.Results: PLEKHA5 was differentially expressed in both the A375 cell line model and patient samples subjected to gene expression profiling. At the protein level, by quantitative immunofluorescence, PLEKHA5 was associated with decreased brain metastasis-free survival. PLEKHA5 overexpression was not associated with other metastatic sites. Knockdown of PLEKHA5 decreases the viability of A375Br cells, inhibits BBB transmigration and invasion in vitro. Similar results were found with YUMUL cells, cultured from a patient with overwhelming brain metastases. PLEKHA5 knockdown did not affect the viability of A375P cells.Conclusions: PLEKHA5 expression in melanoma tumors was associated with early development of brain metastases. Inhibition of PLEKHA5 might decrease passage across the BBB and decrease proliferation and survival of melanoma cells both in the brain and in extracerebral sites. Clin Cancer Res; 21(9); 2138–47. ©2014 AACR.See related commentary by Eisele et al., p. 1978

This file provides legends for Supplementary Figures S1, S2, S3 and S4. Figure S1 contains Kaplan Meier survival curves for PLEKHA5 expression and metastases to various sites, Figures S2 and S3 show Western blots demonstrating PLEKHA5 expression in cells transfected and untransfected with siRNA for PLEKHA5, and Figure S4 shows the effect of astrocytes on PLEKHA5 silencing and cell viability in A375Br cells.

Supplementary Table S3: This table provides the results of a multivariable Cox proportional hazards analysis of PLEKHA5 and other clinical variables. PLEKHA5 was independently associated with development of early brain metastases.

This file provides additional details regarding methods for RNA extraction and quality control, gene expression profiling and analysis, immunofluorescence staining and cell lines used. It also contains information on the siRNA knock-down studies, the in vitro blood brain barrier assay, cell viability and apoptotic assays and the cell motility assay.

Conventional maximum‐tolerated dose (MTD) chemotherapy relies on periodic, massive cancer cell ablation events followed by treatment‐free intermissions, stereotypically resulting in resistance, relapse, and mortality. Furthermore, MTD chemotherapy can promote metastatic dissemination via activation of a transcriptional program dependent on hypoxia‐inducible factor (HIF)‐1α and (HIF)‐2α (hereafter referred to as HIFα). Instead, frequent low‐dose metronomic (LDM) chemotherapy displays less adverse effects while preserving significant pre‐clinical anticancer activity. Consequently, we hereby compared the effect of MTD or LDM chemotherapy upon HIFα in models of advanced, metastatic colon and breast cancer. Our results revealed that LDM chemotherapy could offset paralog‐specific, MTD‐dependent HIFα induction in colon cancers disseminating to the liver and lungs, while limiting HIFα and hypoxia in breast cancer lung metastases. Moreover, we assessed the translational significance of HIFα activity in colorectal and breast TCGA/microarray data, by developing two compact, 11‐gene transcriptomic signatures allowing the stratification/identification of patients likely to benefit from LDM and/or HIFα‐targeting therapies. Altogether, these results suggest LDM chemotherapy as a potential maintenance strategy to stave off HIFα induction within the intra‐metastatic tumor microenvironment., Low‐dose metronomic (LDM) chemotherapy offsets intra‐metastatic tumoral hypoxia, hypoxia‐inducible factor (HIF)‐α induction, and angiogenic responses elicited by conventional maximum‐tolerated dose (MTD) therapies in models of advanced, metastatic colon and breast cancer.

The impressive successes of immune checkpoint blockade antibodies to treat various types of cancer are limited to minor subsets of patients. Combination therapy strategies, including with chemotherapy, are being explored to possibly improve the efficacy of immunotherapies. Here we report results regarding the use of an immunostimulatory regimen of metronomic cyclophosphamide (CTX). We show that in orthotopic models of syngeneic murine triple-negative breast cancer (EMT6), CTX administered at 140 mg/kg every 6 days (CTX140 1q6d) is superior at inhibiting primary tumor growth when compared to maximum tolerated dose or daily oral (continuous) low-dose CTX. In SCID or SCID beige mice, anti-tumor effects of CTX140 1q6d are reduced, reinforcing the therapeutic contribution of the adaptive and innate immune systems. In a second breast cancer model (SP1-AC2M2), CTX140 1q6d again showed clear superiority in anti-tumor effects, causing complete tumor regressions; however, these mice were not protected from subsequent tumor re-challenge, suggesting absence of immune memory. We also show that in an aggressive and metastatic cisplatin-resistant variant (EMT6-CDDP), CTX140 1q6d is superior and invokes an influx of intra-tumoral CD4+ and CD8+ T cells. CTX increases expression of tumor cell PD-L1; however, when combined with concomitant PD-L1 antibody therapy none of the CTX regimens showed increased benefit. This work sheds light on the potential use of metronomic CTX for the treatment of breast cancer, in particular using the quasi-weekly regimen, but also underscores the complexity of the anti-tumor mechanisms and potential to improve immune checkpoint therapy efficacy.

Producción Científica, Metronomic chemotherapy using the 5-FU prodrug uracil-tegafur (UFT) and cyclophosphamide (CTX) was previously shown to only modestly delay primary tumor growth, but nevertheless markedly suppressed the development of micro-metastasis in an orthotopic breast cancer xenograft model, using the metastatic variant of the MDA-MB-231 cell line, 231/LM2-4. Furthermore, a remarkable prolongation of survival, with no toxicity, was observed in a model of postsurgical advanced metastatic disease. A question that has remained unanswered is the seemingly selective anti-metastatic mechanisms of action responsible for this treatment. We assessed the in vivo effect of metronomic UFT, CTX or their combination, on vascular density, collagen deposition and c-Met (cell mediators or modulators of tumor cell invasion or dissemination) via histochemistry/immunohistochemistry of primary tumor sections. We also assessed the effect of continuous exposure to low and non-toxic doses of active drug metabolites 5-fluorouracil (5-FU), 4-hydroperoxycyclophosphamide (4-HC) or their combination, on 231/LM2-4 cell invasiveness in vitro. In the in vivo studies, a significant reduction in vascular density and p-Met[Y1003] levels was associated with UFT+CTX treatment. All treatments reduced intratumoral collagen deposition. In the in vitro studies, a significant reduction of collagen IV invasion by all treatments was observed. The 3D structures formed by 231/LM2-4 on Matrigel showed a predominantly Mass phenotype under treated conditions and Stellate phenotype in untreated cultures. Taken together, the results suggest the low-dose metronomic chemotherapy regimens tested can suppress several mediators of tumor invasiveness highlighting a new perspective for the anti-metastatic efficacy of metronomic chemotherapy., Canadian Institutes of Health Research (grant 364411)

Purpose: Approximately 40% of patients with metastatic melanoma develop brain metastases. Our purpose was to identify genes aberrantly expressed in melanoma that might be associated with propensity for brain homing. Experimental Design: We studied gene expression profiles in a cell line model of brain metastasis (cerebrotropic A375Br cells vs. parental A375P cells) and compared them with profiles of patients who developed early brain metastases and who did not. A tissue microarray containing 169 metastatic melanoma cases with variable time to brain metastasis was constructed to further study marker expression by quantitative immunofluorescence. An in vitro model of the blood brain barrier (BBB) was generated to evaluate potential mediators of brain metastases. Results: PLEKHA5 was differentially expressed in both the A375 cell line model and patient samples subjected to gene expression profiling. At the protein level, by quantitative immunofluorescence, PLEKHA5 was associated with decreased brain metastasis-free survival. PLEKHA5 overexpression was not associated with other metastatic sites. Knockdown of PLEKHA5 decreases the viability of A375Br cells, inhibits BBB transmigration and invasion in vitro. Similar results were found with YUMUL cells, cultured from a patient with overwhelming brain metastases. PLEKHA5 knockdown did not affect the viability of A375P cells. Conclusions: PLEKHA5 expression in melanoma tumors was associated with early development of brain metastases. Inhibition of PLEKHA5 might decrease passage across the BBB and decrease proliferation and survival of melanoma cells both in the brain and in extracerebral sites. Clin Cancer Res; 21(9); 2138–47. ©2014 AACR. See related commentary by Eisele et al., p. 1978

Thousands of cancer patients are currently in clinical trials evaluating antiangiogenic therapy in the neoadjuvant setting, which is the treatment of localized primary tumors prior to surgical intervention. The rationale is that shrinking a tumor will improve surgical outcomes and minimize growth of occult micrometastatic disease— thus delaying post-surgical recurrence and improving survival. But approved VEGF pathway inhibitors have not been tested in clinically relevant neoadjuvant models that compare pre- and post-surgical treatment effects. Using mouse models of breast, kidney, and melanoma metastasis, we demonstrate that primary tumor responses to neoadjuvant VEGFR TKI treatment do not consistently correlate with improved post-surgical survival, with survival worsened in certain settings. Similar negative effects did not extend to protein-based VEGF pathway inhibitors and could be reversed with altered dose, surgical timing, and treatment duration, or when VEGFR TKIs are combined with metronomic ‘anti-metastatic’ chemotherapy regimens. These studies represent the first attempt to recapitulate the complex clinical parameters of neoadjuvant therapy in mice and identify a novel tool to compare systemic antiangiogenic treatment effects on localized and disseminated disease.

The Wnt pathways contribute to many processes in cancer and development, with β-catenin being a key canonical component. p120-catenin, which is structurally similar to β-catenin, regulates the expression of certain Wnt target genes, relieving repression conferred by the POZ- and zinc-finger-domain-containing transcription factor Kaiso. We have identified the kinase Dyrk1A as a component of the p120-catenin–Kaiso trajectory of the Wnt pathway. Using rescue and other approaches in Xenopus laevis embryos and mammalian cells, we found that Dyrk1A positively and selectively modulates p120-catenin protein levels, thus having an impact on p120-catenin and Kaiso (and canonical Wnt) gene targets such as siamois and wnt11. The Dyrk1A gene resides within the Down's syndrome critical region, which is amplified in Down's syndrome. A consensus Dyrk phosphorylation site in p120-catenin was identified, with a mutant mimicking phosphorylation exhibiting the predicted enhanced capacity to promote endogenous Wnt-11 and Siamois expression, and gastrulation defects. In summary, we report the biochemical and functional relationship of Dyrk1A with the p120-catenin–Kaiso signaling trajectory, with a linkage to canonical Wnt target genes. Conceivably, this work might also prove relevant to understanding the contribution of Dyrk1A dosage imbalance in Down's syndrome.

An enduring problem in cancer research is the failure to reproduce highly encouraging preclinical therapeutic findings using transplanted or spontaneous primary tumours in mice in clinical trials of patients with advanced metastatic disease. There are several reasons for this, including the failure to model established, visceral metastatic disease. We therefore developed various models of aggressive multi-organ spontaneous metastasis after surgical resection of orthotopically transplanted human tumour xenografts. In this Opinion article we provide a personal perspective summarizing the prospect of their increased clinical relevance. This includes the reduced efficacy of certain targeted anticancer drugs, the late emergence of spontaneous brain metastases and the clinical trial results evaluating a highly effective therapeutic strategy previously tested using such models.

Purpose: The development of effective therapeutic approaches for treatment of metastatic melanoma remains an immense challenge. Present therapies offer minimal benefit. Although dacarbazine chemotherapy remains the standard therapy, it mediates only low response rates, usually of short duration, even when combined with other chemotherapeutic agents. Thus, new therapeutic strategies are urgently needed. Experimental Design: Using a newly developed preclinical model, we evaluated the efficacy of various doublet metronomic combination chemotherapy against established advanced melanoma metastasis and compared these with the standard maximum tolerated dose dacarbazine (alone or in combination with chemotherapeutic agents or vascular endothelial growth factor receptor–blocking antibody). Results: Whereas maximum tolerated dose dacarbazine therapy did not cause significant improvement in median survival, a doublet combination of low-dose metronomic vinblastine and low-dose metronomic cyclophosphamide induced a significant increase in survival with only minimal toxicity. Furthermore, we show that the incorporation of the low-dose metronomic vinblastine/low-dose metronomic cyclophosphamide combination with a low-dose metronomic dacarbazine regimen also results in a significant increase in survival, but not when combined with maximum tolerated dose dacarbazine therapy. We also show that a combination of metronomic vinblastine therapy and a vascular endothelial growth factor receptor 2–blocking antibody (DC101) results in significant control of metastatic disease and that the combination of low-dose metronomic vinblastine/DC101 and low-dose metronomic dacarbazine induced a significant improvement in median survival. Conclusions: The effective control of advanced metastatic melanoma achieved by these metronomic-based chemotherapeutic approaches warrants clinical consideration of this treatment concept, given the recent results of a number of metronomic-based chemotherapy clinical trials.

SummaryHerein we report that the VEGFR/PDGFR kinase inhibitor sunitinib/SU11248 can accelerate metastatic tumor growth and decrease overall survival in mice receiving short-term therapy in various metastasis assays, including after intravenous injection of tumor cells or after removal of primary orthotopically grown tumors. Acceleration of metastasis was also observed in mice receiving sunitinib prior to intravenous implantation of tumor cells, suggesting possible “metastatic conditioning” in multiple organs. Similar findings with additional VEGF receptor tyrosine kinase inhibitors implicate a class-specific effect for such agents. Importantly, these observations of metastatic acceleration were in contrast to the demonstrable antitumor benefits obtained when the same human breast cancer cells, as well as mouse or human melanoma cells, were grown orthotopically as primary tumors and subjected to identical sunitinib treatments.

Melanoma has a high predilection for neuronal dissemination, with 44% to 64% of patients developing brain metastases (B-Mets) during the course of metastatic progression which represents the leading cause of death in most of these cases (Davies et al., 2011; Johnson et al., 1996; Sampson et al., 1998). Current treatments for melanoma B-Mets aim to slow disease progression and are determined by their number and location (Eichler et al., 2007; Elliott et al., 2011; Rush et al., 2011). Preclinical models of melanoma B-Mets are fundamental for studying the biology behind the dissemination of melanoma cells to the brain and evaluating new therapeutic regimens’ efficacy. Over the years, several groups have reported the development of either experimental or spontaneous models of melanoma B-Mets with the majority using the highly invasive intra-carotid (IC) surgery as a standard technique for tumor induction (Fujimaki et al., 1996; Huang et al., 2008; Xie et al., 2006; Yano et al., 2000; Zhang et al., 2009, Cruz-Munoz et al., 2008). The scarcity of clinically relevant, well characterized models has hindered our understanding of the mechanism of melanoma brain tropism and response to therapies. The need to effectively examine different aspects of metastatic dissemination in vivo has motivated investigators to develop imaging strategies in which MRI is best positioned for intracranial imaging. MRI was used recently in B-Mets murine studies for monitoring the temporal and spatial development of breast lesions (Perera, Ribot et al. 2012). In melanoma preclinical studies, the use of in vivo MRI was limited to the detection of B-Mets (Izraely et al., 2012). Hence, there is still a need to establish the tumor growth profile and pattern of melanoma B-Mets using a 3D quantitative imaging analysis. Here, we aimed to establish an experimental melanoma B-Mets mouse model using the B16F10 murine and the 5B1 human cell lines by means of minimally invasive ultrasound-guided intra-cardiac injection (UGICI). Our goals were to characterize and compare the growth pattern of the developing B-Mets in both models using MRI with micrometric resolution termed micro-MRI (μ-MRI). By utilizing large cohorts of mice (n=80), we intended to overcome the following limitations imposed by in vivo MRI: the slow throughput, the prohibitive cost per scan, as well as limited access to scan time. We opted to use an ex vivo setup devised in our laboratory which enabled simultaneous imaging of multiple whole mouse heads placed in a larger MRI probe. Four heads could be scanned simultaneously during one unattended overnight session (Data S1). Our approach capitalized on the paramagnetic nature of melanin present in both tumor cell lines; the B16F10 murine and to a lesser extent the 5B1 human cell line. The signal brightening endogenous effect of melanoma in tracer-free T1-weighted MRI datasets correlates with the percentage of melanin-containing cells. This feature combined with the signal darkening effect of a T2*-weighted sequence used in our protocol is specific for melanotic tumors but not in amelanotic tumors. Conversely, the latter exhibit T1-weighted hypointense or isointense signal and T2-weighted hyperintense or isointense contrast (Isiklar et al.1995) (Data S2). Our first goal was to establish a safer and physiologically relevant tumor induction route. For that, we used (1×105cells/100μl/mouse) B16F10 cells and C57Bl6 mouse strain (n=18) in each group to compare the safety and efficacy of our proposed UGICI route to the standard IC injection. The UGICI closely simulate the spread of melanoma cells from the blood stream to the brain tissue. Melanoma cells are introduced through the left ventricle allowing them to circulate freely throughout the entire intact vascular system. This provides the cells with an equal opportunity to colonize different organs and thereby objectively reflects inherent tissue tropism. The use of ultrasound to visualize the advancing needle into the left ventricle of the heart in real time reduced the number of puncture attempts and allowed us to monitor and confirm the proper and uniform transmission of cells into each mouse (Data S3). Comparing this novel minimally invasive technique of tumor induction to the traditional IC injection, we noticed a distinct difference in mortality rate ranging from 20% to 25% for the IC to ~1% with the UGICI. Moreover, we recorded a marked decrease in surgery-related morbidity, evident by stable mouse weight reflecting a better ability of mice to mobilize and feed following UGICI (Data S4). Additionally, the time required to induce the animals was significantly shorter using the UGICI (10–12 minutes) compared to the IC (35–60 minutes) allowing for large scale experiments to be conducted, which is essential for achieving meaningful, statistically-sound data. Taking advantage of the safer and more efficient UGICI route for tumor induction, we compared the growth pattern and distribution of two experimental melanoma models based on the B16F10 murine and the 5B1 human cell lines. While the B16F10 has no reported predilection for a particular site of metastasis, the 5B1, an in vivo selected subpopulation of the WM239A cell line, was previously reported to show enhanced brain tropism (Cruz-Munoz et al., 2008). In this system, B16F10 cells showed relatively low brain tropism with 27.5% brain penetrance and high affinity to extra-cranial sites while the 5B1 cells had a high predilection for brain tissue with a penetrance rate of 70% and less extra-cranial organ involvement (Data S5). In addition, the extra-cranial metastatic lesions in the 5B1 model were less aggressive and often unilateral (Data S6). The ex vivo MRI protocol implemented was able to recapitulate the characteristic in vivo clinical radiological findings of melanoma B-Mets in both models: bright enhancing areas on a T1-weighted sequence and susceptibility-induced signal darkening on T2*-weighted imaging (Data S7). These findings are used radiologically to distinguish melanoma B-Mets from other types of B-Mets for which the systemic injection of a contrast agent is usually required in the clinical setting (Gaviani et al., 2006). Although T2*-based imaging did not demonstrate systematic tumor detection via signal darkening, the blooming effect associated with this sequence proved useful in combination with T1-hyperintensity in presence of poor contrast and for identifying small lesions. Hence, the evaluation of the tumor volumes in this study relied principally on T1-weighted imaging in which T2*-darkening was complementary when needed. Ex vivo data acquired from the B16F10 model (n=40) revealed exclusive ventricular and leptomeningeal spread (Data S8a-c), while 5B1 injected mice (n=40) showed generally parenchymal lesions, reflecting the phenotype mostly seen in the clinic (Chen et al., 2012) (Data S8d-f). In addition, MRI allowed for the exploration of the multicentric nature of the two melanoma cell lines used, where 67.9% of the 5B1 injected mice with brain tumors (n=28) showed multiple lesions (range 1 - 16, median: 3) compared to 18% in the B16 mouse model (n=11) (range 1 - 3, median: 1) (Data S9b, c). The 5B1 cells were injected in immune deficient athymic/nude (nu/nu) mice which may account for the higher number of B-Mets developing in the 5B1 model. However, the relatively lower number of extra-cranial metastasis compared to the B16F10 immune competent model suggests that an intrinsically higher brain tropism of the 5B1 cell line, rather than a more permissive host immune system, allows more cells to actively seed and adapt to brain tissue microenvironment. Tumor segmentation, 3D reconstruction and quantitative volumetric analysis at different time points, post-tumor induction (Data S10a for 5B1 model and S10b for B16F10 model) revealed differences in growth pattern of tumors in both models; B16F10 B-Mets were detected on day 15 post-tumor induction at a volume of 0.65 mm3 and exhibited a rapid pattern of growth over a short time frame of 8 days with some lesions detected at a volume of 44.7mm3 on day 23 post-tumor induction (range: 0.14 mm3 - 44.7 mm3, median: 2.01 mm3). The 5B1 metastatic lesions had a delayed onset of tumor detection on day 27 post-injection at a starting volume of 0.03mm3. In addition, the 5B1 lesions were less aggressive and showed a slower growth rate, acquiring a maximum detected volume of 34.01mm3 (range 0.02 mm3 - 34.01 mm3, median: 0.37 mm3) on day 56 post-tumor induction (Data S9a). Using 5B1 melanoma human cell line and UGICI for tumor induction together with our novel ex vivo MRI technique we provide the field with a clinically relevant B-Mets experimental model and a faithful tool to assess changes in tumor behavior in preclinical therapeutic studies.

The spectra of conducting polymers obtained using ultraviolet photoelectron spectroscopy (UPS) exhibit a typical broadening of the tail σUPS ≈ 1 eV, which by an order of magnitude exceeds a commonly accepted value of the broadening of the tail of the density of states σDOS ≈ 0.1 eV obtained using transport measurements. In this work, an origin of this anomalous broadening of the tail of the UPS spectra in a doped conducting polymer, PEDOT (poly(3,4-ethylenedioxythiophene)), is discussed. Based on the semiempirical approach and using a realistic morphological model, the density of valence states in PEDOT doped with molecular counterions is computed. It is shown that due to a disordered character of the material with randomly distributed counterions, the localized charge carriers in PEDOT crystallites experience spatially varying electrostatic potential. This leads to spatially varying local vacuum levels and binding energies. Taking this variation into account the UPS spectrum is obtained with the broadening of the tail comparable to the experimentally observed one. The results imply that the observed broadening of the tail of the UPS spectra in PEDOT provides information about a disordered spatially varying potential in the material rather than the broadening of the DOS itself.

The use of transgenic mouse models as somatic mutation assays allows determination of mutation in all tissues of the mouse, including non-dividing tissues. In this regard, these models can be used to study the possibility that mutations can be induced in mitotically quiescent organs such as the heart. Mutations are generally thought to be associated with mitotic processes of DNA replication. Mutations, however, are also postulated to occur in the absence of mitosis as the result of DNA repair. In order to determine whether or not mutations could be induced in the heart, we analyzed the mutant frequency in the hearts of F(1) (Muta Mouse X SWR) mice that had been treated acutely with 250 mg/kg ENU and sampled at days 10, 35, and 70 post-treatment. A significant increase in mutant frequency at day 70 shows that mutations can be induced in the heart. Since the heart contains small numbers of non-muscle cells, additional mechanisms that could explain these results were also considered. The effect of ENU-induced cell proliferation or a sub-population of rapidly dividing cells is ruled out by C(14)-thymidine uptake studies which showed minimal proliferation. By the same token, the influence of ex vivo mutations (i.e., DNA adducts fixed as mutations during replication in the bacteria) is ruled out by the observed time course of mutations, as well as experimental evidence showing that such mutations are not detected in the lacZ assay.

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta(trade mark)Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue((R)) Mouse. The cII displays similar spontaneous (5.5 x 10(-5)) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N-ethyl-N-nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 x 10(-5) at the cII and 147 x 10(-5) at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R(2) = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays.

An alternative or follow-up adjunct to conventional maximum tolerated dose (MTD) chemotherapy now in advanced phase III clinical trial assessment is metronomic chemotherapy - the close regular administration of low doses of drug with no prolonged breaks. A number of preclinical studies have shown metronomic chemotherapy can cause long term survival of mice with advanced cancer, including metastatic disease, in the absence of overt toxicity, especially when combined with targeted antiangiogenic drugs. However, similar to MTD chemotherapy acquired resistance eventually develops, the basis of which is unknown. Using a preclinical model of advanced human ovarian (SKOV-3-13) cancer in SCID mice, we show that acquired resistance can develop after terminating prolonged (over 3 months) successful therapy utilizing daily oral metronomic topotecan plus pazopanib, an oral antiangiogenic tyrosine kinase inhibitor (TKI). Two resistant sublines were isolated from a single mouse, one from a solid tumor (called KH092-7SD, referred to as 7SD) and another from ascites tumor cells (called KH092-7AS, referred to as 7AS). Using these sublines we show acquired resistance to the combination treatment is due to tumor cell alterations that confer relative refractoriness to topotecan. The resistant phenotype is heritable, associated with reduced cellular uptake of topotecan and could not be reversed by switching to MTD topotecan or to another topoisomerase-1 inhibitor, CPT-11, given either in a metronomic or MTD manner nor switching to another antiangiogenic drug, e.g. The anti-VEGFR-2 antibody, DC101, or another TKI, sunitinib. Thus, in this case cross resistance seems to exist between MTD and metronomic topotecan, the basis of which is unknown. However, gene expression profiling revealed several potential genes that are stably upregulated in the resistant lines, that previously have been implicated in resistance to various chemotherapy drugs, and which, therefore, may contribute to the drug resistant phenotype.

Members of the plakophilin-catenin sub-family (Pkp-1, -2, and -3) facilitate the linkage of desmosome junctional components to each other (e.g. desmosomal cadherins to desmoplakin) and the intermediate-filament cytoskeleton. Pkps also contribute to desmosomal stabilization and the trafficking of its components. The functions of Pkps outside of the desmosome are less well studied, despite evidence suggesting their roles in mRNA regulation, small-GTPase modulation (e.g. mid-body scission) during cell division, and cell survival following DNA damage. Pkp-catenins are further believed to have roles in the nucleus given their nuclear localization in some contexts and the known nuclear roles of structurally related catenins, such as beta-catenin and p120-catenin. Further, Pkp-catenin activities in the nuclear compartment have become of increased interest with the identification of interactions between Pkp2-catenin and RNA Pol III and Pkp1 with single-stranded DNA. Consistent with earlier reports suggesting possible nuclear roles in development, we previously demonstrated prominent nuclear localization of Pkp3 in Xenopus naïve ectoderm ("animal cap") cells and recently resolved a similar localization in mouse embryonic stem cells. Here, we report the association and positive functional interaction of Pkp3 with a transcription factor, Ets variant gene 1 (ETV1), which has critical roles in neural development and prominent roles in human genetic disease. Our results are the first to report the interaction of a sequence-specific transcription factor with any Pkp. Using Xenopus laevis embryos and mammalian cells, we provide evidence for the Pkp3:ETV1 complex on both biochemical and functional levels.

Metastatic spread of melanoma to the central nervous system (CNS) is a common and devastating manifestation of disease progression, which, despite its clinical importance, remains poorly understood with respect to underlying molecular mechanisms. Using a recently developed preclinical model of spontaneous melanoma CNS metastasis, we have identified alterations in expression of endothelin receptor B (EDNRB) as a potential factor that influences brain metastatic potential. Induced overexpression of this gene mediated enhanced overall metastatic disease, and resulted in an increased incidence of spontaneous CNS metastases. In contrast, the overexpression of other highlighted genes, such as BCL2A1, did not affect the incidence of CNS metastases but nevertheless appears to facilitate intracranial tumor growth. The prometastatic effect in the CNS associated with EDNRB appears to be mediated by the interaction with its ligands resulting in enhanced tumor cell proliferation and thus intracranial melanoma growth. That EDNRB contributes to melanoma metastasis is underscored by the fact that its therapeutic inhibition by the EDNRB-specific inhibitor A192621 translated into improved outcomes when treating mice with either visceral metastases or intracranial tumors. The identification of an influential role of EDNRB in CNS melanoma spontaneous metastasis may provide both a target for therapeutic intervention as well as a potential prognostic marker for patients having an increased predisposition for incidence of CNS melanoma metastases. Cancer Res; 72(19); 4909–19. ©2012 AACR.

Metastatic spread to the central nervous system (CNS) is a common and devastating manifestation of major cancer types. Its incidence is associated with poor prognosis manifested by neurological deterioration leading to diminished quality of life and an extremely short median survival. CNS metastasis is becoming an increasingly important clinical problem. This is especially the case for certain types of cancers for which effective treatments of visceral disease are available. As a result of the present limitations in treating CNS metastases, this manifestation of tumor progression remains an unmet clinical need. Despite its significance, our general understanding of the mechanisms that regulate the brain-metastatic phenotype is currently meager. Both the analysis of mechanistic aspects of brain metastasis and the development of effective treatments necessitate the use of appropriate in vivo models that recapitulate the interaction of the tumor cells with the microenvironment of the brain. Here we review the available preclinical models of CNS metastasis and their use as tools to advance knowledge of the biology of the disease (with the aim of identifying relevant molecular determinants, prognostic biomarkers, and therapeutic targets) as well as examining effective approaches for treatment.

The tissue microenvironment impacts health and disease by providing a dynamic media where cells interact with the extracellular matrix scaffold, and a sink for critical ligands that dictate cell function. These dynamics become altered in cancer development through dysregulated proteolysis that is in part controlled by tissue inhibitors of metalloproteinases (TIMPs). This chapter reviews TIMP evolution and structure, the metalloproteinase targets, and the substrate complexity arising from metalloproteinase function. Individually, TIMPs inhibit proteolysis in a temporally and spatially distinct manner, resulting in a variety of phenotypes arising from specific TIMP deficiencies. It also discusses the consequences of altered TIMP gene expression on cell proliferation, apoptosis, angiogenesis, invasion, and metastasis in cell culture systems and mouse models. Although TIMPs inhibit angiogenesis, invasion, and metastasis, their effects on cell proliferation and apoptosis are tissue specific and context dependent. These studies highlight the importance of TIMPs as cancer modifier genes.

Tissue inhibitors of metalloproteinases (TIMPs) are classically known for regulating members of the metzincin protease family and are well recognized for their inhibitory effects in cancer development and progression. Despite their common evolutionary structure, the four TIMP proteins have unique properties and regulation, and produce distinct phenotypes when ablated. A comprehensive assessment of their function during tumorigenesis reveals substantial effects on cell proliferation, apoptosis, angiogenesis, invasion, and metastasis as well as a potential role in genomic instability. The TIMPs universally inhibit angiogenesis, invasion, and metastasis, but their specific effects on cell proliferation and apoptosis are both tissue specific and context dependent. They exert these effects in a metalloproteinase-dependent as well as metalloproteinase-independent manner. Knowledge gained from these biological studies provides a foundation for the full understanding of TIMP function in physiology and various pathologies as well as for the development of the next generation of therapeutic metalloproteinase inhibitors.

Metastatic spread of melanoma to the central nervous system (CNS) is associated with dismal prognosis. Preclinical testing of novel therapeutic approaches would be aided by the development of appropriate models of spontaneous CNS metastasis arising from primary tumors. A highly metastatic variant of the WM239A human melanoma cell line, designated 113/6-4L, was generated and used to test the efficacy of long-term, low-dose metronomic cyclophosphamide and vinblastine chemotherapy on advanced established metastatic disease in sites such as liver, lungs, and lymph node. This treatment resulted in control of advanced, systemic disease and prolongation of survival. Among long-term surviving mice, 20% showed the presence of spontaneous brain metastases. Two cell lines (131/4-5B1 and 131/4-5B2) were generated from such metastases, which were found to spontaneously metastasize to brain parenchyma with occasional localization to leptomeninges, after orthotopic transplantation and removal of the primary tumor. The cell lines were found to have increased ability to proliferate in brain-conditioned medium and displayed enhanced adhesion to lung and brain endothelial cells. These findings represent the first report of spontaneous CNS metastases generated from primary tumors of any human cancer in mice, which heritably maintains this phenotype, and as such, the variant cell lines generated should aid studies in the biology and treatment of CNS metastases, especially of melanoma origin. [Cancer Res 2008;68(12):4500–5]

The functions of Lys(63)-linked polyubiquitin chains are poorly understood, as are the enzymes that specifically generate Lys(63)-linked conjugates. Rsp5 is a HECT (homologous to E6AP C terminus) ubiquitin ligase involved in numerous processes, and an associated deubiquitinating enzyme, Ubp2, modulates its activity. A dramatic increase in Lys(63)-linked conjugates was observed in ubp2Delta cells. The formation of these was Rsp5-dependent, and ubp2Delta phenotypes could be suppressed by prevention of formation of Lys(63) conjugates. Cell wall integrity was impaired in rsp5-1 cells and in cells defective in Lys(63)-polyubiquitination, as assayed by calcofluor white sensitivity, and ubp2Delta and rup1Delta mutants suppressed the calcofluor white sensitivity of rsp5-1. A large fraction of the Lys(63) conjugates in ubp2Delta cells bound to Rsp5, and a proteomics approach was used to identify Rsp5 substrates subject to Ubp2 regulation. Two closely related proteins, Csr2 and Ecm21, were among the identified proteins. Both were efficiently Lys(63)-polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. Together, these results indicate that Ubp2 modulates Lys(63)-polyubiquitination of Rsp5 substrates in vivo, including ubiquitination of two newly identified Rsp5 substrates.

Although a number of Her-2 targeting drugs are now available for the treatment of Her-2 positive breast cancers, resistance to these therapies rapidly emerges. There is therefore a need to study how Her-2 targeting strategies work in vivo, and the mechanisms by which tumors eventually become resistant to anti-Her-2 therapies. We report the in vivo selection of variants of the human Her-2 positive breast cancer cell lines BT474 and MDA-MB361. Both cell lines were initially implanted orthotopically into Severely Compromised Immunodeficient mice, and the resulting tumors were serially passaged into new hosts over a 4 year period. This selection process produced the BT474V3 and MDA361V3 variants, which grow rapidly in vivo and which retain their Her-2 over-expression, and which respond (p Citation Format: Paloma A. Valenzuela, Sarah N. Jallad, Karla Parra, Natzidielly Lerma, Irving Miramontes, Alejandra Gallegos, Ping Xu, William Cruz-Munoz, Shan Man, Robert S. Kerbel, Giulio Francia. Derivation and analysis of preclinical models of human her-2 positive breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3142. doi:10.1158/1538-7445.AM2014-3142

e11061 Background: The anti-Her-2 antibody trastuzumab can be effectively combined with chemotherapy to treat Her-2 positive metastatic breast cancers. However, Her-2 positive breast cancers readily develop resistance to trastuzumab based therapies, and new models are needed to study such resistance, and to find new therapeutic options. Methods: We began the in vivo selection of human Her-2 positive breast cancer cell lines, BT474 and MDA-MB-361, in Severely Compromised Immunodeficient (SCID) mice. Tumor cells were implanted orthotopically into the mammary fat pad of SCID mice, and resulting tumors were serially passaged into new hosts over a 3 year period. Results: The resulting variants grow rapidly in SCID mice. Variants of both BT474 and MDA-MB-361 respond in vivo to trastuzumab monotherapy (20mg/kg twice weekly, i.p.) compared to controls (p

Recent clinical trial failures with antiangiogenic therapy in patients with early-stage disease have raised the possibility that drug efficacy in preclinical studies, typically involving models of localized primary grown tumors, may not always predict for efficacy in treating metastatic disease, which is the most common cause of mortality in patients. Moreover, depending on disease stage and treatment circumstances, the efficacy of VEGF-pathway inhibitors administered as monotherapies may be offset in certain settings by increased aggressive invasiveness and augmented metastatic potential. Thus, with hundreds of trials underway in neoadjuvant and adjuvant settings to evaluate the efficacy of antiangiogenic therapy in blocking or slowing micrometastatic disease and eventual tumor recurrence, there is an urgent need clarify the utility of such drugs in all stages of tumor progression. Herein we describe a novel preclinical neoadjuvant therapeutic methodology used to compare the effects of short-term VEGF pathway inhibition in highly metastatic human breast, melanoma, and kidney tumor cells grown orthotopically in SCID mice before and after surgical resection. While cytotoxic chemotherapy administered in the maximum tolerated dose could slow tumor growth and lead to a prolongation of survival after resection, similar significant benefits by various VEGF inhibitors in primary tumor inhibition did not consistently translate into significant prevention of recurrent local and distant metastasis after treatment cessation. Furthermore, reductions in primary tumor growth by neutralizing antibodies to VEGF or VEGFR-2 were more predictive of modest survival benefits after tumor resection, compared to the VEGFR tyrosine kinase inhibitor (TKI) sunitinib, which had either no effect or decreased survival compared to control - despite significant reductions in primary tumor growth after neoadjuvant treatment. Potential tumor-independent ‘host’ drug responses could account for this difference as short-term treatment of immunocompromised nu/nu mice prior to intravenous tumor inoculation lead to modest or negligible benefits with VEGF or VEGFR2 antibodies compared to worse outcomes in VEGFR TKI pre-treated animals. Taken together, preclinical neoadjuvant therapy allows for the distinction of ‘anti-primary’ and ‘anti-metastatic’ effects and may serve as a predictive tool to model ongoing clinical trials as well as indicate potential drug combinations which may be used to overcome limitations when such drugs are used as a monotherapy. 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 1375. doi:1538-7445.AM2012-1375

Low dose metronomic (LDM) chemotherapy has shown promising activity in many preclinical as well as some phase II clinical studies involving various tumor types. It involves chronic but close, regular administration of chemotherapeutic drugs without prolonged breaks. Combination of LDM chemotherapy with targeted biologic agents such as antiangiogenic drugs can sometimes induce striking anti-tumor effects in preclinical models, with minimal toxicity. The most encouraging clinical results thus far involve patients with metastatic breast cancer and recurrent metastatic ovarian cancer. Regarding the ovarian cancer trials, all have involved daily low-dose oral cyclophosphamide using a fixed dose of 50mg, combined with bevacizumab. However, there may be much more effective chemotherapy drugs for LDM chemotherapy-based treatments of ovarian cancer. Here we evaluated the impact of optimal biologic doses (OBDs) of LDM oral topotecan, a topo-isomerase-1 inhibitor already approved for refractory ovarian cancer, in combination with oral pazopanib, a receptor tyrosine kinase inhibitor which targets VEGF and PDGF receptors. Clones of the SKOV-3 human ovarian carcinoma cell line expressing secretable β-subunit of human choriogonadotropic (β-hCG) protein and firefly luciferase were generated, and evaluated for growth after orthotopic (intraperitoneal) injection into immune deficient SCID mice, and a highly aggressive clone, SKOV-3-13, was selected for further study. Treatments were initiated 14 days after injection of 3×106 cells when evidence of carcinomatosis-like disease in the peritoneum was established as assessed by imaging analysis. The drug concentrations were 1mg/kg for oral topotecan by daily gavage, 150mg/kg pazopanib daily gavage or 25mg/kg twice/day, and combinations of oral topotecan+pazopanib. These doses were based on suppressive effects on circulating endothelial progenitor cells, a method we have used previously to determine the OBD for other LDM chemotherapy regimens. Metronomic oral topotecan showed excellent anti-tumor activity, the extent of which was significantly enhanced by concurrent pazopanib, which itself had modest activity. The median survival value of mice in the control group was 34 days after start of treatment compared with 73 days for MTD topotecan, 41 days for 25mg/kg pazopanib, 41 days for 150mg/kg pazopanib, 90 days for oral topotecan, whereas all of the mice in the two oral topotecan/pazopanib combination treatment groups were still alive 150 days after initiation of treatment. Overt toxic side effect was not observed throughout the treatment period. Metronomic topotecan in combination with an antiangiogenic VEGF-pathway targeting drug, such as pazopanib should be considered for evaluation in clinical trials involving patients with advanced, recurrent epithelial ovarian cancer. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A9.

In Response: Dr. Hoffman is correct to highlight his previous work showing detection of spontaneous human melanoma brain metastases by using imaging-based techniques. Indeed, we cited one of his articles ( [1][1]) mentioned in his letter as reference 16 in our Clinical Cancer Research article. We