Aaron K. Mobley

A Survey on Data Reproducibility in Cancer Research Provides Insights into Our Limited Ability to Translate Findings from the Laboratory to the Clinic

Background The pharmaceutical and biotechnology industries depend on findings from academic investigators prior to initiating programs to develop new diagnostic and therapeutic agents to benefit cancer patients. The success of these programs depends on the validity of published findings. This validity, represented by the reproducibility of published findings, has come into question recently as investigators from companies have raised the issue of poor reproducibility of published results from academic laboratories. Furthermore, retraction rates in high impact journals are climbing. Methods and Findings To examine a microcosm of the academic experience with data reproducibility, we surveyed the faculty and trainees at MD Anderson Cancer Center using an anonymous computerized questionnaire; we sought to ascertain the frequency and potential causes of non-reproducible data. We found that ∼50% of respondents had experienced at least one episode of the inability to reproduce published data; many who pursued this issue with the original authors were never able to identify the reason for the lack of reproducibility; some were even met with a less than “collegial” interaction. Conclusions These results suggest that the problem of data reproducibility is real. Biomedical science needs to establish processes to decrease the problem and adjudicate discrepancies in findings when they are discovered.

Galectin-3 Contributes to Melanoma Growth and Metastasis via Regulation of NFAT1 and Autotaxin

Melanoma is the deadliest form of skin cancer in which patients with metastatic disease have a 5-year survival rate of less than 10%. Recently, the overexpression of a β-galactoside binding protein, galectin-3 (LGALS3), has been correlated with metastatic melanoma in patients. We have previously shown that silencing galectin-3 in metastatic melanoma cells reduces tumor growth and metastasis. Gene expression profiling identified the protumorigenic gene autotaxin (ENPP2) to be downregulated after silencing galectin-3. Here we report that galectin-3 regulates autotaxin expression at the transcriptional level by modulating the expression of the transcription factor NFAT1 (NFATC2). Silencing galectin-3 reduced NFAT1 protein expression, which resulted in decreased autotaxin expression and activity. Reexpression of autotaxin in galectin-3 silenced melanoma cells rescues angiogenesis, tumor growth, and metastasis in vivo. Silencing NFAT1 expression in metastatic melanoma cells inhibited tumor growth and metastatic capabilities in vivo. Our data elucidate a previously unidentified mechanism by which galectin-3 regulates autotaxin and assign a novel role for NFAT1 during melanoma progression.

Why is melanoma so metastatic

Malignant melanoma is one of the most aggressive cancers and can disseminate from a relatively small primary tumor and metastasize to multiple sites, including the lung, liver, brain, bone, and lymph nodes. Elucidating the molecular and genetic changes that take place during the metastatic process has led to a better understanding of why melanoma is so metastatic. Herein, we describe the unique features that distinguish melanoma from other solid tumors and contribute to the malignant phenotype of melanoma cells. For example, although melanoma cells are highly antigenic, they are extremely efficient at evading host immune response. Melanoma cells share numerous cell surface molecules with vascular cells, are highly angiogenic, are mesenchymal in nature, and possess a higher degree of ‘stemness’ than do other solid tumors. Finally, analysis of melanoma mutations has revealed that the gene expression profile of malignant melanoma is different from that of other cancers. Elucidating these molecular and genetic processes in highly metastatic melanoma can lead to the development of improved treatment and individualized therapy options.

A mosaic mouse model of astrocytoma identifies alphavbeta8 integrin as a negative regulator of tumor angiogenesis.

Angiogenesis involves a complex set of cell–cell and cell–extracellular matrix (ECM) interactions that coordinately promote and inhibit blood vessel growth and sprouting. Although many factors that promote angiogenesis have been characterized, the identities and mechanisms of action of endogenous inhibitors of angiogenesis remain unclear. Furthermore, little is known about how cancer cells selectively circumvent the actions of these inhibitors to promote pathological angiogenesis, a requisite event for tumor progression. Using mosaic mouse models of the malignant brain cancer, astrocytoma, we report that tumor cells induce pathological angiogenesis by suppressing expression of the ECM protein receptor αvβ8 integrin. Diminished integrin expression in astrocytoma cells leads to reduced activation of latent TGFβs, resulting in impaired TGFβ receptor signaling in tumor-associated endothelial cells. These data reveal that astrocytoma cells manipulate their angiogenic balance by selectively suppressing αvβ8 integrin expression and function. Finally, these results show that an adhesion and signaling axis normally involved in developmental brain angiogenesis is pathologically exploited in adult brain tumors.

β8 integrin is essential for neuroblast migration in the rostral migratory stream

Neurogenesis in the post‐natal brain occurs in two primary locations: the subgranular layer of the hippocampal dentate gyrus and the subventricular zone (SVZ) of the lateral ventricles. Following differentiation, neuroblasts within the SVZ migrate several millimeters to the olfactory bulbs (OBs) via a distinct anatomic route, or rostral migratory stream (RMS). The genes that govern neuroblast directional migration, and particularly those encoding cell adhesion and signaling factors, remain largely uncharacterized. Here, we report that the extracellular matrix adhesion receptor, β8 integrin, is essential for proper neuroblast chain formation and directional navigation in the RMS. Primary neuroblasts isolated from the mouse brain express robust levels of β8 integrin protein, and selective ablation of β8 integrin gene expression in neuroblasts leads to aberrant chain migration and size‐reduced OBs. These integrin‐dependent defects can be recapitulated ex vivo using isolated neurospheres or SVZ explants. Collectively, these data identify essential cell‐intrinsic functions for β8 integrin in regulating neuroblast polarity and directional navigation in the mouse forebrain.

A mosaic mouse model of astrocytoma identifies αvβ8 integrin as a negative regulator of tumor angiogenesis

Angiogenesis involves a complex set of cell–cell and cell–extracellular matrix (ECM) interactions that coordinately promote and inhibit blood vessel growth and sprouting. Although many factors that promote angiogenesis have been characterized, the identities and mechanisms of action of endogenous inhibitors of angiogenesis remain unclear. Furthermore, little is known about how cancer cells selectively circumvent the actions of these inhibitors to promote pathological angiogenesis, a requisite event for tumor progression. Using mosaic mouse models of the malignant brain cancer, astrocytoma, we report that tumor cells induce pathological angiogenesis by suppressing expression of the ECM protein receptor αvβ8 integrin. Diminished integrin expression in astrocytoma cells leads to reduced activation of latent TGFβs, resulting in impaired TGFβ receptor signaling in tumor-associated endothelial cells. These data reveal that astrocytoma cells manipulate their angiogenic balance by selectively suppressing αvβ8 integrin expression and function. Finally, these results show that an adhesion and signaling axis normally involved in developmental brain angiogenesis is pathologically exploited in adult brain tumors.

NFAT1 Directly Regulates IL8 and MMP3 to Promote Melanoma Tumor Growth and Metastasis

Nuclear factor of activated T cell (NFAT1, NFATC2) is a transcription factor that binds and positively regulates IL2 expression during T-cell activation. NFAT1 has important roles in both innate and adaptive immune responses, but its involvement in cancer is not completely understood. We previously demonstrated that NFAT1 contributes to melanoma growth and metastasis by regulating the autotaxin gene (Enpp2). Here, we report a strong correlation between NFAT1 expression and metastatic potential in melanoma cell lines and tumor specimens. To elucidate the mechanisms underlying NFAT1 overexpression during melanoma progression, we conducted a microarray on a highly metastatic melanoma cell line in which NFAT1 expression was stably silenced. We identified and validated two downstream targets of NFAT1, IL8, and MMP3. Accordingly, NFAT1 depletion in metastatic melanoma cell lines was associated with reduced IL8 and MMP3 expression, whereas NFAT1 overexpression in a weakly metastatic cell line induced expression of these targets. Restoration of NFAT1 expression recovered IL8 and MMP3 expression levels back to baseline, indicating that both are direct targets of NFAT1. Moreover, in vivo studies demonstrated that NFAT1 and MMP3 promoted melanoma tumor growth and lung metastasis. Collectively, our findings assign a new role for NFAT1 in melanoma progression, underscoring the multifaceted functions that immunomodulatory factors may acquire in an unpredictable tumor microenvironment. Cancer Res; 76(11); 3145-55. ©2016 AACR.

Aurora Kinase A is a Biomarker for Bladder Cancer Detection and Contributes to its Aggressive Behavior

The effects of AURKA overexpression associated with poor clinical outcomes have been attributed to increased cell cycle progression and the development of genomic instability with aneuploidy. We used RNA interference to examine the effects of AURKA overexpression in human bladder cancer cells. Knockdown had minimal effects on cell proliferation but blocked tumor cell invasion. Whole genome mRNA expression profiling identified nicotinamide N-methyltransferase (NNMT) as a downstream target that was repressed by AURKA. Chromatin immunoprecipitation and NNMT promoter luciferase assays revealed that AURKA’s effects on NNMT were caused by PAX3-mediated transcriptional repression and overexpression of NNMT blocked tumor cell invasion in vitro. Overexpression of AURKA and activation of its downstream pathway was enriched in the basal subtype in primary human tumors and was associated with poor clinical outcomes. We also show that the FISH test for the AURKA gene copy number in urine yielded a specificity of 79.7% (95% confidence interval [CI] = 74.2% to 84.1%), and a sensitivity of 79.6% (95% CI = 74.2% to 84.1%) with an AUC of 0.901 (95% CI = 0.872 to 0.928; P < 0.001). These results implicate AURKA as an effective biomarker for bladder cancer detection as well as therapeutic target especially for its basal type.

Crossing the junction in the gap of melanoma brain metastasis

Instead, the authors revealed that NRP1 promoted the migration of regulatory T cells toward VEGF both in vitro and in vivo. Primary melanomas developing in mice that lack NRP1 in T cells showed impaired infiltration with Foxp3+ regulatory T cells. Adoptive transfer of NRP1+ regulatory T cells from wild-type mice was able to reverse this phenotype. Thus, genetic blockade of NRP1 in T cells decreased VEGF-dependent recruitment of regulatory T cells into the tumor microenvironment and thereby reinforced local antitumor immunity leading to reduced tumor growth and prolonged tumor-free survival. The elegant experiments performed by Hansen et al. ask more questions than they answer. In a mouse model of experimental autoimmune encephalitis, it was shown that conditional deletion of NRP1 in T cells led to the preferential induction of Th-17 cells in addition to decreased regulatory T cell function. In this experimental system, NRP1expressing CD4+ T cells suppressed effector T-cell proliferation and cytokine production both in vivo and in vitro independent of regulatory T cells but dependent on TGF-b. Future studies will have to address the role of CD4+ T cells and of TGF-b also in the melanoma model, as both play critically important roles in the control of cellular antitumor immunity. Furthermore, the molecular mechanisms by which NRP1 regulates the migration of regulatory T cells remain to be determined. NRP1 might well engage semaphorins which have also been shown to be functionally expressed by immune cells. Yet unknown mechanism might also be discovered, given the binding of NRPs with multiple ligand families that orchestrate its diverse biological functions beyond neural and vascular development (Raimondi and Ruhrberg, 2013). An increasing number of reports show that NRP1 also participates in tumor angiogenesis. Experiments with tumor xenografts in immunodeficient mice showed a modest suppressive effect of anti-NRP1 antibodies on tumor growth when given alone, but demonstrated significant additive suppressive effects when applied in combination with anti-VEGF antibodies. This resulted in the reduction in tumor vessel density and maturity. The data provided a scientific basis for clinical trials targeting NRP1 as an anti-angiogenic therapeutic strategy that could overcome the resistance to anti-VEGF therapies. Studying the effect of anti-NRP1 mAbs on the interaction between tumor and immune cells alone or in combination with antiVEGF mAbs in an immunocompetent experimental mouse model would be highly informative in the future. Recent studies indicate that NRPs are also highly expressed in diverse tumor cell lines and in human tumor tissue, including melanoma. Expression of NRPs has also been linked to a poor prognosis in several tumor entities. This is consistent with their interactions with ligands and receptors that promote tumor progression. As NRP1 helps to guide the migration of neural crest cell precursors, it is conceivable that NRP1 also mediates melanoma metastasis. Therapeutic blockade of NRP1 might therefore beneficially modulate multiple protumorigenic processes operative in melanoma progression.

Use of Cre-Lox Technology to Analyze Integrin Functions in Astrocytes

Astrocytes communicate with the vascular endothelium via direct cell-cell contacts as well as a variety of secreted growth factors and extracellular matrix (ECM) proteins. Integrins are heterodimeric cell surface receptors for ECM protein ligands, and many integrin subunits are expressed in astrocytes. Here, we will discuss gene deletion strategies in mice that have deciphered functions for specific integrins in astrocyte-endothelial cell adhesion and signaling. Specifically, we will detail how Cre-lox molecular genetic techniques have revealed important roles for integrin αvβ8 in regulating cerebral blood vessel development and homeostasis. First, we will detail how to generate Cre-lox mutant mouse models that our group and others have used to study αvβ8 integrin in embryonic astroglial progenitors and postnatal astrocytes. Second, we will discuss how viral-delivered Cre can be used to acutely delete integrin genes in astrocytes within defined anatomic regions of the brain. Third, detailed in vivo methods to verify Cre-mediated gene recombination in astrocytes will be presented. Lastly, we will present one experimental strategy to determine how integrin gene deletion affects astrocyte-endothelial cell coupling in the CNS. While this review focuses on the generation and characterization of mice lacking αvβ8 integrin, these experimental strategies can be expanded to analyze other cell adhesion and signaling genes important for astroglial-mediated regulation of blood vessel development and homeostasis.