Characterization of two different alginate-based bioinks and the influence of melanoma growth within.

Extrusion-based bioprinting is an established method in biofabrication. Suitable bioinks have fundamentally different compositions and characteristics, which should be examined, in order to find a perfect model system. Here, we investigate the effect of two alginate-based, yet unalike 3D-printed bioinks, pre-crosslinked alginate-dialdehyde gelatin (ADA-GEL) and a mixture of alginate, hyaluronic acid, and gelatin (Alg/HA/Gel), on the melanoma cell line Mel Im and vice versa in terms of stiffness, shrinkage, cellular behavior and colony formation over 15 days. Rheological stiffness measurements revealed two soft gels with similar storage moduli. The cells did not have a significant impact on the overall stiffness, whereas ADA-GEL (2.5/2.5%) was significantly stiffer than Alg/HA/Gel (0.5/0.1/3%). Regarding the shrinkage of printed constructs, cells had a significant influence, especially in ADA-GEL, which has covalent bonds between the oxidized alginate and gelatin. Multi-photon microscopy exhibited proliferation, cell spreading and migration in ADA-GEL with cell-cell and cell-matrix interaction, dissimilarly to Alg/HA/Gel, in which cells formed spherical, encapsulated colonies. Scanning electron microscopy and histology showed degradation and multi-layered growth on ADA-GEL and fewer examples of escaped cells on Alg/HA/Gel. Both gels serve as proliferation bioink for melanoma with more necrosis in deeper Alg/HA/Gel colonies and differences in spreading and matrix interaction. These findings show the importance of proper characterization of the bioinks for different applications.

A vascularized in vivo melanoma model suitable for metastasis research of different tumor stages using fundamentally different bioinks.

Although 2D cancer models have been the standard for drug development, they don't resemble in vivo properties adequately. 3D models can potentially overcome this. Bioprinting is a promising technique for more refined models to investigate central processes in tumor development such as proliferation, dormancy or metastasis. We aimed to analyze bioinks, which could mimic these different tumor stages in a cast vascularized arteriovenous loop melanoma model in vivo. It has the advantage to be a closed system with a defined microenvironment, supplied only with one vessel-ideal for metastasis research. Tested bioinks showed significant differences in composition, printability, stiffness and microscopic pore structure, which led to different tumor stages (Matrigel and Alg/HA/Gel for progression, Cellink Bioink for dormancy) and resulted in different primary tumor growth (Matrigel significantly higher than Cellink Bioink). Light-sheet fluorescence microscopy revealed differences in vascularization and hemorrhages with no additional vessels found in Cellink Bioink. Histologically, typical human melanoma with different stages was demonstrated. HMB-45-positive tumors in progression inks were infiltrated by macrophages (CD163), highly proliferative (Ki67) and metastatic (MITF/BRN2, ATX, MMP3). Stainings of lymph nodes revealed metastases even without significant primary tumor growth in Cellink Bioink. This model can be used to study tumor pathology and metastasis of different tumor stages and therapies.

Pirin is a prognostic marker of human melanoma that dampens the proliferation of malignant cells by downregulating JARID1B/KDM5B expression.

Originally considered to act as a transcriptional co-factor, Pirin has recently been reported to play a role in tumorigenesis and the malignant progression of many tumors. Here, we have analyzed the diagnostic and prognostic value of Pirin expression in the early stages of melanoma, and its role in the biology of melanocytic cells. Pirin expression was analyzed in a total of 314 melanoma biopsies, correlating this feature with the patient's clinical course. Moreover, PIR downregulated primary melanocytes were analyzed by RNA sequencing, and the data obtained were validated in human melanoma cell lines overexpressing PIR by functional assays. The immunohistochemistry multivariate analysis revealed that early melanomas with stronger Pirin expression were more than twice as likely to develop metastases during the follow-up. Transcriptome analysis of PIR downregulated melanocytes showed a dampening of genes involved in the G1/S transition, cell proliferation, and cell migration. In addition, an in silico approach predicted that JARID1B as a potential transcriptional regulator that lies between PIR and its downstream modulated genes, which was corroborated by co-transfection experiments and functional analysis. Together, the data obtained indicated that Pirin could be a useful marker for the metastatic progression of melanoma and that it participates in the proliferation of melanoma cells by regulating the slow-cycling JARID1B gene.

Sox9 regulates melanocytic fate decision of adult hair follicle stem cells.

The bulge of hair follicles harbors Nestin+ (neural crest like) stem cells, which exhibit the potential to generate various cell types including melanocytes. In this study, we aimed to determine the role of Sox9, an important regulator during neural crest development, in melanocytic differentiation of those adult Nestin+ cells. Immunohistochemical analysis after conditional Sox9 deletion in Nestin+ cells of adult mice revealed that Sox9 is crucial for melanocytic differentiation of these cells and that Sox9 acts as a fate determinant between melanocytic and glial fate. A deeper understanding of factors that regulate fate decision, proliferation and differentiation of these stem cells provides new aspects to melanoma research as melanoma cells share many similarities with neural crest cells. In summary, we here show the important role of Sox9 in melanocytic versus glial fate decision of Nestin+ stem cells in the skin of adult mice.

A previously unknown Argonaute 2 variant positively modulates the viability of melanoma cells.

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.

Multifrequency Investigation of Single- and Double-Stranded DNA with Scalable Metamaterial-Based THz Biosensors.

Due to the occurrence of THz-excited vibrational modes in biomacromolecules, the THz frequency range has been identified as particularly suitable for developing and applying new bioanalytical methods. We present a scalable THz metamaterial-based biosensor being utilized for the multifrequency investigation of single- and double-stranded DNA (ssDNA and dsDNA) samples. It is demonstrated that the metamaterial resonance frequency shift by the DNA's presence depends on frequency. Our experiments with the scalable THz biosensors demonstrate a major change in the degree of the power function for dsDNA by 1.53 ± 0.06 and, in comparison, 0.34 ± 0.11 for ssDNA as a function of metamaterial resonance frequency. Thus, there is a significant advantage for dsDNA detection that can be used for increased sensitivity of biomolecular detection at higher frequencies. This work represents a first step for application-specific biosensors with potential advantages in sensitivity, specificity, and robustness.

Alpha-Synuclein and Its Role in Melanocytes.

Pigmentation is an important process in skin physiology and skin diseases and presumably also plays a role in Parkinson's disease (PD). In PD, alpha-Synuclein (aSyn) has been shown to be involved in the pigmentation of neurons. The presynaptic protein is intensively investigated for its pathological role in PD, but its physiological function remains unknown. We hypothesized that aSyn is both involved in melanocytic differentiation and melanosome trafficking processes. We detected a strong expression of aSyn in human epidermal melanocytes (NHEMs) and observed its regulation in melanocytic differentiation via the microphthalmia-associated transcription factor (MITF), a central regulator of differentiation. Moreover, we investigated its role in pigmentation by performing siRNA experiments but found no effect on the total melanin content. We discovered a localization of aSyn to melanosomes, and further analysis of aSyn knockdown revealed an important role in melanocytic morphology and a reduction in melanosome release. Additionally, we found a reduction of transferred melanosomes in co-culture experiments of melanocytes and keratinocytes but no complete inhibition of melanosome transmission. In summary, this study highlights a novel physiological role of aSyn in melanocytic morphology and its so far unknown function in the pigment secretion in melanocytes.

3D hydrogel-based microcapsules as an in vitro model to study tumorigenicity, cell migration and drug resistance.

In this work, we analyzed the reliability of alginate-gelatin microcapsules as artificial tumor model. These tumor-like scaffolds are characterized by their composition and stiffness (∼25 kPa), and their capability to restrict -but not hinder- cell migration, proliferation and release from confinement. Hydrogel-based microcapsules were initially utilized to detect differences in mechano-sensitivity between MCF7 and MDA-MB-231 breast cancer cells, and the endothelial cell line EA.hy926. Additionally, we used RNA-seq and transcriptomic methods to determine how the culture strategy (i.e. 2D v/s 3D) may pre-set the expression of genes involved in multidrug resistance, being then validated by performing cytotoxicological tests and assays of cell morphology. Our results show that both breast cancer cells can generate elongated multicellular spheroids inside the microcapsules, prior being released (mimicking intravasation stages), a behavior which was not observed in endothelial cells. Further, we demonstrate that cells isolated from 3D scaffolds show resistance to cisplatin, a process which seems to be strongly influenced by mechanical stress, instead of hypoxia. We finally discuss the role played by aneuploidy in malignancy and resistance to anticancer drugs, based on the increased number of polynucleated cells found within these microcapsules. Overall, our outcomes demonstrate that alginate-gelatin microcapsules represent a simple, yet very accurate tumor-like model, enabling us to mimic the most relevant malignant hints described in vivo, suggesting that confinement and mechanical stress need to be considered when studying pathogenicity and drug resistance of cancer cells in vitro. STATEMENT OF SIGNIFICANCE: In this work, we analyzed the reliability of alginate-gelatin microcapsules as an artificial tumor model. These scaffolds are characterized by their composition, elastic properties, and their ability to restrict cell migration, proliferation, and release from confinement. Our results demonstrate four novel outcomes: (i) studying cell migration and proliferation in 3D enabled discrimination between malignant and non-pathogenic cells, (ii) studying the cell morphology of cancer aggregates entrapped in alginate-gelatin microcapsules enabled determination of malignancy degree in vitro, (iii) determination that confinement and mechanical stress, instead of hypoxia, are required to generate clones resistant to anticancer drugs (i.e. cisplatin), and (iv) evidence that resistance to anticancer drugs could be due to the presence of polynucleated cells localized inside polymer-based artificial tumors.

HDAC2 Is Involved in the Regulation of BRN3A in Melanocytes and Melanoma.

The neural crest transcription factor BRN3A is essential for the proliferation and survival of melanoma cells. It is frequently expressed in melanoma but not in normal melanocytes or benign nevi. The mechanisms underlying the aberrant expression of BRN3A are unknown. Here, we investigated the epigenetic regulation of BRN3A in melanocytes and melanoma cell lines treated with DNA methyltransferase (DNMT), histone acetyltransferase (HAT), and histone deacetylase (HDAC) inhibitors. DNMT and HAT inhibition did not significantly alter BRN3A expression levels, whereas panHDAC inhibition by trichostatin A led to increased expression. Treatment with the isoform-specific HDAC inhibitor mocetinostat, but not with PCI-34051, also increased BRN3A expression levels, suggesting that class I HDACs HDAC1, HDAC2, and HDAC3, and class IV HDAC11, were involved in the regulation of BRN3A expression. Transient silencing of HDACs 1, 2, 3, and 11 by siRNAs revealed that, specifically, HDAC2 inhibition was able to increase BRN3A expression. ChIP-Seq analysis uncovered that HDAC2 inhibition specifically increased H3K27ac levels at a distal enhancer region of the BRN3A gene. Altogether, our data suggest that HDAC2 is a key epigenetic regulator of BRN3A in melanocytes and melanoma cells. These results highlight the importance of epigenetic mechanisms in regulating melanoma oncogenes.

Loss of Gene Information: Discrepancies between RNA Sequencing, cDNA Microarray, and qRT-PCR.

Molecular analyses of normal and diseased cells give insight into changes in gene expression and help in understanding the background of pathophysiological processes. Years after cDNA microarrays were established in research, RNA sequencing (RNA-seq) became a key method of quantitatively measuring the transcriptome. In this study, we compared the detection of genes by each of the transcriptome analysis methods: cDNA array, quantitative RT-PCR, and RNA-seq. As expected, we found differences in the gene expression profiles of the aforementioned techniques. Here, we present selected genes that exemplarily demonstrate the observed differences and calculations to reveal that a strong RNA secondary structure, as well as sample preparation, can affect RNA-seq. In summary, this study addresses an important issue with a strong impact on gene expression analysis in general. Therefore, we suggest that these findings need to be considered when dealing with data from transcriptome analyses.

Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels.

Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.

3D Spheroids Versus 3D Tumor-Like Microcapsules: Confinement and Mechanical Stress May Lead to the Expression of Malignant Responses in Cancer Cells.

As 2D surfaces fail to resemble the tumoral milieu, current discussions are focused on which 3D cell culture strategy may better lead the cells to express in vitro most of the malignant hints described in vivo. In this study, this question is assessed by analyzing the full genetic profile of MCF7 cells cultured either as 3D spheroids-considered as "gold standard" for in vitro cancer research- or immobilized in 3D tumor-like microcapsules, by RNA-Seq and transcriptomic methods, allowing to discriminate at big-data scale, which in vitro strategy can better resemble most of the malignant features described in neoplastic diseases. The results clearly show that mechanical stress, rather than 3D morphology only, stimulates most of the biological processes involved in cancer pathogenicity, such as cytoskeletal organization, migration, and stemness. Furthermore, cells entrapped in hydrogel-based scaffolds are likely expressing other physiological hints described in malignancy, such as the upregulated expression of metalloproteinases or the resistance to anticancer drugs, among others. According to the knowledge, this study represents the first attempt to answer which 3D experimental system can better mimic the neoplastic architecture in vitro, emphasizing the relevance of confinement in cancer pathogenicity, which can be easily achieved by using hydrogel-based matrices.

Combined De-Repression of Chemoresistance Associated Mitogen-Activated Protein Kinase 14 and Activating Transcription Factor 2 by Loss of microRNA-622 in Hepatocellular Carcinoma.

Chemoresistance is a major hallmark driving the progression and poor prognosis of hepatocellular carcinoma (HCC). Limited chemoresponse of HCC was demonstrated to be mediated by mitogen-activated protein kinase 14 (MAPK14) and activating transcription factor 2 (ATF2). Recently, we have demonstrated loss of control of RAS-RAF-ERK-signaling as a consequence of miR-622 downregulation in HCC. However, the majority of target genes of this potent tumorsuppressive microRNA had remained elusive. The MAPK14-ATF2-axis represents a collateral pathway ensuring persisting ERK-activation in the presence of sorafenib-mediated RAF-inhibition. In contrast to the function of the MAPK14-ATF2-axis, both the expression and regulation of MAPK14 and ATF2 in human HCC remained to be clarified. We found combined overexpression of MAPK14 and ATF2 in human HCC cells, tissues and in sorafenib resistant cell lines. High expression of MAPK14 and ATF2 was associated with reduced overall survival in HCC patients. Deciphering the molecular mechanism promoting combined upregulation of MAPK14 and ATF2 in HCC, we revealed that miR-622 directly targets both genes, resulting in combined de-repression of the MAPK14-ATF2-axis. Together, miR-622 represents a superior regulator of both RAS-RAF-ERK as well as MAPK14-ATF2-signaling pathways in liver cancer.

Identification of novel targets of miR-622 in hepatocellular carcinoma reveals common regulation of cooperating genes and outlines the oncogenic role of zinc finger CCHC-type containing 11.

The poor prognosis of advanced hepatocellular carcinoma (HCC) is driven by diverse features including dysregulated microRNAs inducing drug resistance and stemness. Lin-28 homolog A (LIN28A) and its partner zinc finger CCHC-type containing 11 (ZCCHC11) cooperate in binding, oligouridylation and subsequent degradation of tumorsuppressive let-7 precursor microRNAs. Functionally, activation of LIN28A was recently shown to promote stemness and chemoresistance in HCC. However, the expression and regulation of LIN28A in HCC had been unclear. Moreover, the expression, regulation and function of ZCCHC11 in liver cancer remained elusive. In contrast to "one-microRNA-one-target" interactions, we identified common binding sites for miR-622 in both LIN28A and ZCCHC11, suggesting miR-622 to function as a superior pathway regulator. Applying comprehensive microRNA database screening, human hepatocytes and HCC cell lines, patient-derived tissue samples as well as "The Cancer Genome Atlas" (TCGA) patient cohorts, we demonstrated that loss of tumorsuppressive miR-622 mediates derepression and overexpression of LIN28A in HCC. Moreover, the cooperator of LIN28A, ZCCHC11, was newly identified as a prognostic and therapeutic target of miR-622 in liver cancer. Together, identification of novel miR-622 target genes revealed common regulation of cooperating genes and outlines the previously unknown oncogenic role of ZCCHC11 in liver cancer.

Amphiregulin Regulates Melanocytic Senescence.

Oncogene-induced senescence (OIS) is a decisive process to suppress tumor development, but the molecular details of OIS are still under investigation. Using an established OIS model of primary melanocytes transduced with BRAF V600E and compared to control cells, amphiregulin (AREG) was shown to be induced. In addition, AREG expression was observed in nevi, which by definition, are senescent cell clusters, compared to melanocytes. Interestingly, treatment of melanocytes with recombinant AREG did induce senescence. This led to the assumption that extracellular AREG has an important function in this process. Inhibition of the epidermal growth factor receptor (EGFR) using Gefitinib identified AREG as one of EGFR ligands responsible for senescence. Furthermore, depletion of AREG expression in senescent BRAF V600E melanocytes resulted in a significant reduction of senescent melanocytes. This study reveals AREG as an essential molecular component of signaling pathways leading to senescence in melanocytes.

Comparison of Hydrogels for the Development of Well-Defined 3D Cancer Models of Breast Cancer and Melanoma.

Bioprinting offers the opportunity to fabricate precise 3D tumor models to study tumor pathophysiology and progression. However, the choice of the bioink used is important. In this study, cell behavior was studied in three mechanically and biologically different hydrogels (alginate, alginate dialdehyde crosslinked with gelatin (ADA-GEL), and thiol-modified hyaluronan (HA-SH crosslinked with PEGDA)) with cells from breast cancer (MDA-MB-231 and MCF-7) and melanoma (Mel Im and MV3), by analyzing survival, growth, and the amount of metabolically active, living cells via WST-8 labeling. Material characteristics were analyzed by dynamic mechanical analysis. Cell lines revealed significantly increased cell numbers in low-percentage alginate and HA-SH from day 1 to 14, while only Mel Im also revealed an increase in ADA-GEL. MCF-7 showed a preference for 1% alginate. Melanoma cells tended to proliferate better in ADA-GEL and HA-SH than mammary carcinoma cells. In 1% alginate, breast cancer cells showed equally good proliferation compared to melanoma cell lines. A smaller area was colonized in high-percentage alginate-based hydrogels. Moreover, 3% alginate was the stiffest material, and 2.5% ADA-GEL was the softest material. The other hydrogels were in the same range in between. Therefore, cellular responses were not only stiffness-dependent. With 1% alginate and HA-SH, we identified matrices that enable proliferation of all tested tumor cell lines while maintaining expected tumor heterogeneity. By adapting hydrogels, differences could be accentuated. This opens up the possibility of understanding and analyzing tumor heterogeneity by biofabrication.

RKIP Regulates Differentiation-Related Features in Melanocytic Cells.

Raf Kinase Inhibitor Protein (RKIP) has been extensively reported as an inhibitor of key signaling pathways involved in the aggressive tumor phenotype and shows decreased expression in several types of cancers. However, little is known about RKIP in melanoma or regarding its function in normal cells. We examined the role of RKIP in both primary melanocytes and malignant melanoma cells and evaluated its diagnostic and prognostic value. IHC analysis revealed a significantly higher expression of RKIP in nevi compared with early-stage (stage I-II, AJCC 8th) melanoma biopsies. Proliferation, wound healing, and collagen-coated transwell assays uncovered the implication of RKIP on the motility but not on the proliferative capacity of melanoma cells as RKIP protein levels were inversely correlated with the migration capacity of both primary and metastatic melanoma cells but did not alter other parameters. As shown by RNA sequencing, endogenous RKIP knockdown in primary melanocytes triggered the deregulation of cellular differentiation-related processes, including genes (i.e., ZEB1, THY-1) closely related to the EMT. Interestingly, NANOG was identified as a putative transcriptional regulator of many of the deregulated genes, and RKIP was able to decrease the activation of the NANOG promoter. As a whole, our data support the utility of RKIP as a diagnostic marker for early-stage melanomas. In addition, these findings indicate its participation in the maintenance of a differentiated state of melanocytic cells by modulating genes intimately linked to the cellular motility and explain the progressive decrease of RKIP often described in tumors.

Ultrasensitive THz biosensor for PCR-free cDNA detection based on frequency selective surfaces.

THz technologies are a powerful tool for label-free detection of biomolecules. However, significant reduction of the lower detection limit is required to apply THz-sensors in biomedical diagnosis. This paper reports an ultrasensitive THz-biosensor based on asymmetric double split ring resonators (aDSRR) for the direct label- and PCR-free detection of DNA at physiologically relevant concentrations. We introduce selective functionalization and localized electric field concentration to enhance aDSRR sensitivity and specificity. The sensor characteristics are demonstrated using the human tumor marker MIA in cDNA samples produced from total RNA without PCR-amplification. Measurements of DNA samples with concentrations as low as 1.55 × 10-12 mol/l are presented.

Dissimilar Appearances Are Deceptive-Common microRNAs and Therapeutic Strategies in Liver Cancer and Melanoma.

: In this review, we summarize the current knowledge on miRNAs as therapeutic targets in two cancer types that were frequently described to be driven by miRNAs-melanoma and hepatocellular carcinoma (HCC). By focusing on common microRNAs and associated pathways in these-at first sight-dissimilar cancer types, we aim at revealing similar molecular mechanisms that are evolved in microRNA-biology to drive cancer progression. Thereby, we also want to outlay potential novel therapeutic strategies. After providing a brief introduction to general miRNA biology and basic information about HCC and melanoma, this review depicts prominent examples of potent oncomiRs and tumor-suppressor miRNAs, which have been proven to drive diverse cancer types including melanoma and HCC. To develop and apply miRNA-based therapeutics for cancer treatment in the future, it is essential to understand how miRNA dysregulation evolves during malignant transformation. Therefore, we highlight important aspects such as genetic alterations, miRNA editing and transcriptional regulation based on concrete examples. Furthermore, we expand our illustration by focusing on miRNA-associated proteins as well as other regulators of miRNAs which could also provide therapeutic targets. Finally, design and delivery strategies of miRNA-associated therapeutic agents as well as potential drawbacks are discussed to address the question of how miRNAs might contribute to cancer therapy in the future.

Molecular crosstalk between Y5 receptor and neuropeptide Y drives liver cancer.

Hepatocellular carcinoma (HCC) is clearly age-related and represents one of the deadliest cancer types worldwide. As a result of globally increasing risk factors including metabolic disorders, the incidence rates of HCC are still rising. However, the molecular hallmarks of HCC remain poorly understood. Neuropeptide Y (NPY) and NPY receptors represent a highly conserved, stress-activated system involved in diverse cancer-related hallmarks including aging and metabolic alterations, but its impact on liver cancer had been unclear. Here, we observed increased expression of NPY5 receptor (Y5R) in HCC, which correlated with tumor growth and survival. Furthermore, we found that its ligand NPY was secreted by peritumorous hepatocytes. Hepatocyte-derived NPY promoted HCC progression by Y5R activation. TGF-ß1 was identified as a regulator of NPY in hepatocytes and induced Y5R in invasive cancer cells. Moreover, NPY conversion by dipeptidylpeptidase 4 (DPP4) augmented Y5R activation and function in liver cancer. The TGF-ß/NPY/Y5R axis and DPP4 represent attractive therapeutic targets for controlling liver cancer progression.