Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose–response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.

As a member of the family of A Disintegrin And Metalloproteinases (ADAM) ADAM8 is preferentially expressed in lymphatic organs, immune cells, and tumor cells. The substrate spectrum for ADAM8 proteolytic activity is not exclusive but is related to effectors of inflammation and signaling in the tumor microenvironment. In addition, complexes of ADAM8 with extracellular binding partners such as integrin β-1 cause an extensive intracellular signaling in tumor cells, thereby activating kinase pathways with STAT3, ERK1/2, and Akt signaling, which causes increased cell survival and enhanced motility. The cytoplasmic domain of ADAM8 harbors five SRC homology-3 (SH3) domains that can potentially interact with several proteins involved in actin dynamics and cell motility, including Myosin 1F (MYO1F), which is essential for neutrophil motility. The concept of ADAM8 thus involves immune cell recruitment, in most cases leading to an enhancement of inflammatory (asthma, COPD) and tumor (including pancreatic and breast cancers) pathologies. In this review, we report on available studies that qualify ADAM8 as a therapeutic target in different pathologies. As a signaling hub, ADAM8 controls extracellular, intracellular, and intercellular communication, the latter one mainly mediated by the release of extracellular vesicles with ADAM8 as cargo. Here, we will dissect the contribution of different domains to these distinct ways of communication in several pathologies. We conclude that therapeutic targeting attempts for ADAM8 should consider blocking more than a single domain and that this requires a thorough evaluation of potent molecules targeting ADAM8 in an in vivo setting.

BACKGROUND:Approximately 35% of pituitary adenoma (PA) display an aggressive profile, resulting in low surgical total resection rates, high recurrence rates, and worse prognosis. However, the molecular mechanism of PA invasion remains poorly understood. Although "a disintegrin and metalloproteinases" (ADAMs) are associated with the progression of many tumors, there are no reports on ADAM22 in PA. METHODS:PA transcriptomics databases and clinical specimens were used to analyze the expression of ADAM22. PA cell lines overexpressing wild-type ADAM22, the point mutation ADAM22, the mutated ADAM22 without disintegrin domain, and knocking down ADAM22 were generated. Cell proliferation/invasion assays, flow cytometry, immunohistochemistry, immunofluorescence, co-immunoprecipitation, mass spectrometry, Reverse transcription-quantitative real-time PCR, phos-tag SDS-PAGE, and Western blot were performed for function and mechanism research. Nude mice xenograft models and rat prolactinoma orthotopic models were used to validate in vitro findings. RESULTS:ADAM22 was significantly overexpressed in PA and could promote the proliferation, migration, and invasion of PA cells. ADAM22 interacted with integrin β1 (ITGB1) and activated FAK/PI3K and FAK/ERK signaling pathways through its disintegrin domain to promote PA progression. ADAM22 was phosphorylated by PKA and recruited 14-3-3, thereby delaying its degradation. ITGB1-targeted inhibitor (anti-itgb1) exerted antitumor effects and synergistic effects in combination with somatostatin analogs or dopamine agonists in treating PA. CONCLUSIONS:ADAM22 was upregulated in PA and was able to promote PA proliferation, migration, and invasion by activating ITGB1 signaling. PKA may regulate the degradation of ADAM22 through post-transcriptional modification levels. ITGB1 may be a potential therapeutic target for PA.

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite multimodal therapy, median survival is poor at 12-15 months. At the molecular level, radio-/chemoresistance and resulting tumor progression are attributed to a small fraction of tumor cells, termed glioblastoma stem-like cells (GSCs). These CD133-expressing, self-renewing cells display the properties of multi-lineage differentiation, resulting in the heterogenous composition of GBM. MicroRNAs (miRNAs) as regulators of gene expression at the post-transcriptional level can alter many pathways pivotal to cancer stem cell fate. This study explored changes in the miRNA expression profiles in patient-derived GSCs altered on differentiation into glial fiber acid protein (GFAP)-expressing, astrocytic tumor cells using a polymerase chain reaction (PCR) array. Initially, 22 miRNAs showed higher expression in GSCs and 9 miRNAs in differentiated cells. The two most downregulated miRNAs in differentiated GSCs were miR-17-5p and miR-425-5p, whilst the most upregulated miRNAs were miR-223-3p and let-7-5p. Among those, miR-425-5p showed the highest consistency in an upregulation in all three GSCs. By transfection of a 425-5p miRNA mimic, we demonstrated downregulation of the GFAP protein in differentiated patient-derived GBM cells, providing potential evidence for direct regulation of miRNAs in the GSC/GBM cell transition.

The metalloprotease-disintegrin ADAM8 is critically involved in the progression of pancreatic cancer. Under malignant conditions, ADAM8 is highly expressed and could play an important role in cell–cell communication as expression has been observed in tumor and immune cells of the tumor microenvironment (TME) such as macrophages. To analyze the potential role of ADAM8 in the TME, ADAM8 knockout PDAC tumor cells were generated, and their release of extracellular vesicles (EVs) was analyzed. In EVs, ADAM8 is present as an active protease and associated with lipocalin 2 (LCN2) and matrix metalloprotease 9 (MMP-9) in an ADAM8-dependent manner, as ADAM8 KO cells show a lower abundance of LCN2 and MMP-9. Sorting of ADAM8 occurs independent of TSG101, even though ADAM8 contains the recognition motif PTAP for the ESCRTI protein TSG101 within the cytoplasmic domain (CD). When tumor cells were co-cultured with macrophages (THP-1 cells), expression of LCN2 and MMP-9 in ADAM8 KO cells was induced, suggesting that macrophage signaling can overcome ADAM8-dependent intracellular signaling in PDAC cells. In co-culture with macrophages, regulation of MMP-9 is independent of the M1/M2 polarization state, whereas LCN2 expression is preferentially affected by M1-like macrophages. From these data, we conclude that ADAM8 has a systemic effect in the tumor microenvironment, and its expression in distinct cell types has to be considered for ADAM8 targeting in tumors.

About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.

As the second most common primary intracranial neoplasms, about 40% of pituitary adenomas (PAs) exhibit aggressive behaviors and resulting in poor patient prognosis. The molecular mechanisms underlying the aggressive behaviors of PAs are not yet fully understood. Biochemical studies have reported that programmed cell death 10 (PDCD10) is a component of the striatin-interacting phosphatase and kinase (STRIPAK) complex and plays a dual role in cancers in a tissue- or disease-specific manner. In the present study, we report for the first time that the role of PDCD10 in PAs. Cell proliferation, migration and invasion were either enhanced by overexpressing or inhibited by silencing PDCD10 in PA cells. Moreover, PDCD10 significantly promoted epithelial-mesenchymal transition (EMT) of pituitary adenoma cells. Mechanistically, we showed that the expression of CXCR2, together with phosphorylation levels of AKT and ERK1/2 were regulated by PDCD10. Activation of CXCR2 inversed inactivation of AKT/ERK signal pathways and the tumor-suppressive effects induced by PDCD10 silencing. Finally, the pro-oncogenic effect of PDCD10 was confirmed by in vivo tumor grafting. Taken together, we demonstrate for the first time that PDCD10 can induce aggressive behaviors of PAs by promoting cellular proliferation, migration, invasion and EMT through CXCR2-AKT/ERK signaling axis.

Current standard adjuvant therapy of glioblastoma multiforme (GBM) using temozolomide (TMZ) frequently fails due to therapy resistance. Thus, novel therapeutic approaches are highly demanded. We tested the therapeutic efficacy of the second-generation XPO1 inhibitor Eltanexor using assays for cell viability and apoptosis in GBM cell lines and GBM stem-like cells. For most GBM-derived cells, IC50 concentrations for Eltanexor were below 100 nM. In correlation with reduced cell viability, apoptosis rates were significantly increased. GBM stem-like cells presented a combinatorial effect of Eltanexor with TMZ on cell viability. Furthermore, pretreatment of GBM cell lines with Eltanexor significantly enhanced radiosensitivity in vitro. To explore the mechanism of apoptosis induction by Eltanexor, TP53-dependent genes were analyzed at the mRNA and protein level. Eltanexor caused induction of TP53-related genes, TP53i3, PUMA, CDKN1A, and PML on both mRNA and protein level. Immunofluorescence of GBM cell lines treated with Eltanexor revealed a strong accumulation of CDKN1A, and, to a lesser extent, of p53 and Tp53i3 in cell nuclei as a plausible mechanism for Eltanexor-induced apoptosis. From these data, we conclude that monotherapy with Eltanexor effectively induces apoptosis in GBM cells and can be combined with current adjuvant therapies to provide a more effective therapy of GBM.

Glioblastoma, as the most aggressive brain tumor, is associated with a poor prognosis and outcome. To optimize prognosis and clinical therapy decisions, there is an urgent need to stratify patients with increased risk for recurrent tumors and low therapeutic success to optimize individual treatment. Radiogenomics establishes a link between radiological and pathological information. This review provides a state-of-the-art picture illustrating the latest developments in the use of radiogenomic markers regarding prognosis and their potential for monitoring recurrence. Databases PubMed, Google Scholar, and Cochrane Library were searched. Inclusion criteria were defined as diagnosis of glioblastoma with histopathological and radiological follow-up. Out of 321 reviewed articles, 43 articles met these inclusion criteria. Included studies were analyzed for the frequency of radiological and molecular tumor markers whereby radiogenomic associations were analyzed. Six main associations were described: radiogenomic prognosis, MGMT status, IDH, EGFR status, molecular subgroups, and tumor location. Prospective studies analyzing prognostic features of glioblastoma together with radiological features are lacking. By reviewing the progress in the development of radiogenomic markers, we provide insights into the potential efficacy of such an approach for clinical routine use eventually enabling early identification of glioblastoma recurrence and therefore supporting a further personalized monitoring and treatment strategy.

Pituitary adenomas (PAs) are mostly benign endocrine tumors that can be treated by resection or medication. However, up to 10% of PAs show an aggressive behavior with invasion of adjacent tissue, rapid proliferation, or recurrence. Here, we provide an overview of target structures in aggressive PAs and summarize current clinical trials including, but not limited to, PAs. Mainly, drug targets in PAs are based on general features of tumor cells such as immune checkpoints, so that programmed cell death 1 (ligand 1) (PD-1/PD-L1) targeting may bear potential to cure aggressive PAs. In addition, epidermal growth factor receptor (EGFR), mammalian target of rapamycin (mTOR), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and their downstream pathways are triggered in PAs, thereby modulating tumor cell proliferation, migration and/or tumor angiogenesis. Temozolomide (TMZ) can be an effective treatment of aggressive PAs. Combination of TMZ with 5-Fluorouracil (5-FU) or with radiotherapy could strengthen the therapeutic effects as compared to TMZ alone. Dopamine agonists (DAs) are the first line treatment for prolactinomas. Dopamine receptors are also expressed in other subtypes of PAs which renders Das potentially suitable to treat other subtypes of PAs. Furthermore, targeting the invasive behavior of PAs could improve therapy. In this regard, human matrix metalloproteinase (MMP) family members and estrogens receptors (ERs) are highly expressed in aggressive PAs, and numerous studies demonstrated the role of these proteins to modulate invasiveness of PAs. This leaves a number of treatment options for aggressive PAs as reviewed here.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such as matrix metalloproteinases (MMPs) and membrane-anchored metalloproteinase disintegrins (ADAMs). Here, we observed using intravital microscopy that Adam8-/- mice had impaired neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic inhibition of ADAM8 attenuated neutrophil infiltration and lung injury while improving bacterial containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired proteolysis but resulted from reduced intracellular interactions of ADAM8 with the actin-based motor molecule Myosin1f that suppressed neutrophil motility. In 2 ARDS cohorts, we analyzed lung fluid proteolytic signatures and identified that ADAM8 activity was positively correlated with disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

Acute respiratory distress syndrome (ARDS) results in catastrophic lung failure and has an urgent, unmet need for improved early recognition and therapeutic development. Neutrophil influx is a hallmark of ARDS and is associated with the release of tissue-destructive immune effectors, such neutrophil transmigration. In mouse pneumonia models, both genetic deletion and pharmacologic containment. Unexpectedly, the alterations of neutrophil function were not attributable to impaired disease severity. We propose that in acute inflammatory lung diseases such as pneumonia and ARDS, ADAM8 inhibition might allow fine-tuning of neutrophil responses for therapeutic gain.

Characterization of brain tumours requires neuropathological expertise and is generally performed by histological evaluation and molecular analysis. One emerging technique to assist pathologists in future tumour diagnostics is multimodal optical spectroscopy. In the current clinical routine, tissue preprocessing with formalin is widely established and suitable for spectroscopic investigations since degradation processes impede the measurement of native tissue. However, formalin fixation results in alterations of the tissue chemistry and morphology for example by protein cross-linking. As optical spectroscopy is sensitive to these variations, we evaluate the effects of formalin fixation on multimodal brain tumour data in this proof-of-concept study. Nonfixed and formalin-fixed cross sections of different common human brain tumours were subjected to analysis of chemical variations using ultraviolet and Fourier-transform infrared microspectroscopy. Morphological changes were assessed by elastic light scattering microspectroscopy in the visible wavelength range. Data were analysed with multivariate data analysis and compared with histopathology. Tissue type classifications deduced by optical spectroscopy are highly comparable and independent from the preparation and the fixation protocol. However, formalin fixation leads to slightly better classification models due to improved stability of the tissue. As a consequence, spectroscopic methods represent an appropriate additional contrast for chemical and morphological information in neuropathological diagnosis and should be investigated to a greater extent. Furthermore, they can be included in the clinical workflow even after formalin fixation.

Due to a grim prognosis, there is an urgent need to detect pancreatic ductal adenocarcinoma (PDAC) prior to metastasis. However, reliable diagnostic imaging methods or biomarkers for PDAC or its precursor lesions are still scarce. ADAM8, a metalloprotease-disintegrin, is highly expressed in PDAC tissue and negatively correlates with patient survival. The aim of our study was to determine the ability of ADAM8-positive extracellular vesicles (EVs) and cargo microRNAs (miRNAs) to discriminate precursor lesions or PDAC from healthy controls. In order to investigate enrichment of ADAM8 on EVs, these were isolated from serum of patients with PDAC ( n = 52), precursor lesions ( n = 7) and healthy individuals ( n = 20). Nanoparticle Tracking Analysis and electron microscopy indicated successful preparation of EVs that were analyzed for ADAM8 by FACS. Additionally, EV cargo analyses of miRNAs from the same serum samples revealed the presence of miR-720 and miR-451 by qPCR and was validated in 20 additional PDAC samples. Statistical analyses included Wilcoxon rank test and ROC curves. FACS analysis detected significant enrichment of ADAM8 in EVs from patients with PDAC or precursor lesions compared to healthy individuals ( p = 0.0005). ADAM8-dependent co-variates, miR-451 and miR-720 were also diagnostic, as patients with PDAC had significantly higher serum levels of miR-451 and lower serum levels of miR-720 than healthy controls and reached high sensitivity and specificity (AUC = 0.93 and 1.00, respectively) to discriminate PDAC from healthy control. Thus, detection of ADAM8-positive EVs and related cargo miR-720 and miR-451 may constitute a specific biomarker set for screening individuals at risk for PDAC.

The metalloproteinase ADAM8 is upregulated in several cancers but has a dispensable function under physiological conditions. In tumor cells, ADAM8 is involved in invasion, migration, and angiogenesis. The use of bivalent inhibitors could impair migration and invasion through the double binding to a homodimeric form of ADAM8 located on the cell surface of tumor cells. Herein we report the rational design and synthesis of the first dimeric ADAM8 inhibitors selective over ADAM10 and matrix metalloproteinases. Bivalent derivatives have been obtained by dimerizing the structure of a previously described ADAM17 inhibitor, JG26. In particular, derivative 2 was shown to inhibit ADAM8 proteolytic activity in vitro and in cell-based assays at nanomolar concentration. Moreover, it was more effective than the parent monomeric compound in blocking invasiveness in the breast cancer MDA-MB-231 cell line, thus supporting our hypothesis about the importance of inhibiting the active homodimer of ADAM8.

Metastatic breast cancer is one of the most common causes of cancer-related death in women worldwide. The transmembrane metalloprotease-disintegrin (ADAM8) protein is highly overexpressed in triple-negative breast cancer (TNBC) cells and potentiates tumor cell invasion and extracellular matrix remodeling. Exploiting the high expression levels of ADAM8 in TNBC cells by delivering anti-ADAM8 antibodies efficiently to the targeted site can be a promising strategy for therapy of TNBC. For instance, a targeted approach with the aid of ultra-high field magnetic resonance imaging (UHF-MRI) activatable thermosensitive liposomes (Lip(TS-GD)) could specifically increase the intracellular accumulation of cytotoxic drugs. The surface of doxorubicin-loaded Lip(TS-GD) was modified by covalent coupling of MAB1031 antibody (Lip(TS-GD-MAB)) in order to target the overexpressed ADAM8 in ADAM8 positive MDA-MB-231 cells. Physicochemical characterization of these liposomes was performed using size, surface morphology and UHF-MRI imaging analysis. In vitro cell targeting was investigated by the washing and circulation method. Intracellular trafficking and lysosomal colocalization were assessed by fluorescence microscopy. Cell viability, biocompatibility and in-ovo CAM assays were performed to determine the effectiveness and safety profiles of liposome formulations. Our results show specific binding and induction of doxorubicin release after Lip(TS-GD-MAB) treatment caused a higher cytotoxic effect at the cellular target site.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer type with one of the highest mortalities. The metalloprotease-disintegrin ADAM8 is highly expressed in pancreatic cancer cells and is correlated with an unfavorable patient prognosis. However, no information is available on ADAM8 expression in cells of the tumor microenvironment. We used immunohistochemistry (IHC) to describe the stromal cell types expressing ADAM8 in PDAC patients using a cohort of 72 PDAC patients. We found ADAM8 expressed significantly in macrophages (6%), natural killer cells (40%), and neutrophils (63%), which showed the highest percentage of ADAM8 expressing stromal cells. We quantified the amount of ADAM8+ neutrophils in post-capillary venules in PDAC sections by IHC. Notably, the amount of ADAM8+ neutrophils could be correlated with post-operative patient survival times. In contrast, neither the total neutrophil count in peripheral blood nor the neutrophil-to-lymphocyte ratio showed a comparable correlation. We conclude from our data that ADAM8 is, in addition to high expression levels in tumor cells, present in tumor-associated stromal macrophages, NK cells, and neutrophils and, in addition to functional implications, the ADAM8-expressing neutrophil density in post-capillary venules is a diagnostic parameter for PDAC patients when the numbers of ADAM8+ neutrophils are quantified.

Senescence is a cellular process that can be initiated by certain stressors such as UVA irradiation. The mechanism by which skin cells protect themselves from the UVA-induced senescence has not been fully investigated. Here, we demonstrate that Bach2 modulates the extent of UVA-induced photoaging through regulation of autophagy in skin fibroblasts. In fact chronic exposure of skin fibroblasts to UVA resulted in a significant decrease in Bach2 expression, both in vitro and in vivo. In addition, knockdown of Bach2 in skin fibroblasts led to an increased expression of cell senescence-related genes, which further enhanced the UVA irradiation-induced photoaging. On the other hand, the overexpression of Bach2 resulted in a decrease in the expression of cell senescence-related genes. We also demonstrate that the knockdown of Bach2 in skin fibroblasts can lead to a decreased expression of autophagy-related genes and vice versa, suggesting that autophagy is involved in Bach2-mediated regulation of senescence in skin fibroblasts. Additionally, inhibition of autophagy with autophagy inhibitor 3-MA suppressed the expression of autophagy-related proteins and promoted cell senescence. Furthermore, knockout of Atg5 or Atg7 in embryonic mouse fibroblasts led to a significant increase in the expression of cell senescencerelated genes. Immunoprecipitation assays further demonstrated that Bach2 directly interacts with Beclin-1, Atg3, Atg7, and LC3 in fibroblasts. Taken together, these findings revealed a critical role for Bach2 in suppressing the UVA irradiation-induced cell senescence via autophagy in skin fibroblasts. Bach2 can therefore be a potential target for the therapy of UV-induced photoaging because of its ability to regulate the process of autophagy in the skin.

Hepatocellular carcinoma (HCC) is one of the most common metastatic tumours. Tumour growth and metastasis depend on the induction of cell proliferation and migration by various mediators. Here, we report that the A Disintegrin and Metalloproteinase (ADAM) 8 is highly expressed in murine HCC tissues as well as in murine and human hepatoma cell lines Hepa1-6 and HepG2, respectively. To establish a dose-dependent role of different ADAM8 expression levels for HCC progression, ADAM8 expression was either reduced via shRNA- or siRNA-mediated knockdown or increased by using a retroviral overexpression vector. These two complementary approaches revealed that ADAM8 expression levels correlated positively with proliferation, clonogenicity, migration and matrix invasion and negatively with apoptosis of hepatoma cells. Furthermore, the analysis of pro-migratory and proliferative signalling pathways revealed that ADAM8 expression level was positively associated with expression of β1 integrin as well as with the activation of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), Src kinase and Rho A GTPase. Finally, up-regulation of promigatory signalling and cell migration was also seen with a proteolytically inactive ADAM8 mutant. These findings reveal that ADAM8 is critically up-regulated in hepatoma cells contributes to cell proliferation and survival and furthermore induces pro-migratory signalling pathways independently of its proteolytic activity. By this, ADAM8 can promote cell functions most relevant for HCC growth and metastasis.