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Abstract Background There has been a rise in endometrial cancer (EC) incidence leading to increased mortality. To counter this trend, improving the stratification of post-surgery recurrence risk and anticipating disease relapse and treatment resistance is essential. Liquid biopsy analyses offer a promising tool for these clinical challenges, though the best strategy for applying them in EC must be defined. This study was designed to determine the value of cfDNA/ctDNA monitoring in improving the clinical management of patients with localized and recurrent disease. Methods Plasma samples and uterine aspirates (UA) from 198 EC patients were collected at surgery and over time. The genetic landscape of UAs was characterized using targeted sequencing. Total cfDNA was analyzed for ctDNA presence based on the UA mutational profile. Results High cfDNA levels and detectable ctDNA at baseline correlated with poor prognosis for DFS (p-value

Chromosomal instability (CIN) is a hallmark of cancer aggressiveness, providing genetic plasticity and tumor heterogeneity that allows the tumor to evolve and adapt to stress conditions. CIN is considered a cancer therapeutic biomarker because healthy cells do not exhibit CIN. Despite recent efforts to identify therapeutic strategies related to CIN, the results obtained have been very limited. CIN is characterized by a genetic signature where a collection of genes, mostly mitotic regulators, are overexpressed in CIN‐positive tumors, providing aggressiveness and poor prognosis. We attempted to identify new therapeutic strategies related to CIN genes by performing a drug screen, using cells that individually express CIN‐associated genes in an inducible manner. We find that the overexpression of targeting protein for Xklp2 (TPX2) enhances sensitivity to the proto‐oncogene c‐Src (SRC) inhibitor dasatinib due to activation of the Yes‐associated protein 1 (YAP) pathway. Furthermore, using breast cancer data from The Cancer Genome Atlas (TCGA) and a cohort of cancer‐derived patient samples, we find that both TPX2 overexpression and YAP activation are present in a significant percentage of cancer tumor samples and are associated with poor prognosis; therefore, they are putative biomarkers for selection for dasatinib therapy.

Abstract Glioblastoma (GB) is a devastating tumor of the central nervous system characterized by a poor prognosis. One of the best-established predictive biomarker in IDH-wildtype GB is O6-methylguanine-DNA methyltransferase (MGMT) methylation (mMGMT), which is associated with improved treatment response and survival. However, current efforts to monitor GB patients through mMGMT detection have proven unsuccessful. Small extracellular vesicles (sEVs) hold potential as a key element that could revolutionize clinical practice by offering new possibilities for liquid biopsy. This study aimed to determine the utility of sEV-based liquid biopsy as a predictive biomarker and disease monitoring tool in patients with IDH-wildtype GB. Our findings show consistent results with tissue-based analysis, achieving a remarkable sensitivity of 85.7% for detecting mMGMT in liquid biopsy, the highest reported to date. Moreover, we suggested that liquid biopsy assessment of sEV-DNA could be a powerful tool for monitoring disease progression in IDH-wildtype GB patients. This study highlights the critical significance of overcoming molecular underdetection, which can lead to missed treatment opportunities and misdiagnoses, possibly resulting in ineffective therapies. The outcomes of our research significantly contribute to the field of sEV-DNA-based liquid biopsy, providing valuable insights into tumor tissue heterogeneity and establishing it as a promising tool for detecting GB biomarkers. These results have substantial implications for advancing predictive and therapeutic approaches in the context of GB and warrant further exploration and validation in clinical settings.

Abstract Mammary gland development occurs primarily in adulthood, undergoing extensive expansion during puberty followed by cycles of functional specialization and regression with every round of pregnancy/lactation/involution. This process is ultimately driven by the coordinated proliferation and differentiation of mammary epithelial cells. However, the endogenous molecular factors regulating these developmental dynamics are still poorly defined. Endocannabinoid signaling is known to determine cell fate-related events during the development of different organs in the central nervous system and the periphery. Here, we report that the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) plays a pivotal role in adult mammary gland development. Specifically, it is required for luminal lineage specification in the mammary gland, and it promotes hormone-driven secretory differentiation of mammary epithelial cells by controlling the endogenous levels of anandamide and the subsequent activation of cannabinoid CB1 receptors. Together, our findings shed light on the role of the endocannabinoid system in breast development and point to FAAH as a therapeutic target in milk-production deficits.

Abstract Clinical management of breast cancer (BC) metastasis remains an unmet need as it accounts for 90% of BC-associated mortality. Although the luminal subtype, which represents >70% of BC cases, is generally associated with a favorable outcome, it is susceptible to metastatic relapse as late as 15 years after treatment discontinuation. Seeking therapeutic approaches as well as screening tools to properly identify those patients with a higher risk of recurrence is therefore essential. Here, we report that the lipid-degrading enzyme fatty acid amide hydrolase (FAAH) is a predictor of long-term survival in patients with luminal BC, and that it blocks tumor progression and lung metastasis in cell and mouse models of BC. Together, our findings highlight the potential of FAAH as a biomarker with prognostic value in luminal BC and as a therapeutic target in metastatic disease.

Abstract Background Gasdermin B (GSDMB) over-expression promotes poor prognosis and aggressive behavior in HER2 breast cancer by increasing resistance to therapy. Decoding the molecular mechanism of GSDMB-mediated drug resistance is crucial to identify novel effective targeted treatments for HER2/GSDMB aggressive tumors. Methods Different in vitro approaches (immunoblot, qRT-PCR, flow cytometry, proteomic analysis, immunoprecipitation, and confocal/electron microscopy) were performed in HER2 breast and gastroesophageal carcinoma cell models. Results were then validated using in vivo preclinical animal models and analyzing human breast and gastric cancer samples. Results GSDMB up-regulation renders HER2 cancer cells more resistant to anti-HER2 agents by promoting protective autophagy. Accordingly, the combination of lapatinib with the autophagy inhibitor chloroquine increases the therapeutic response of GSDMB-positive cancers in vitro and in zebrafish and mice tumor xenograft in vivo models. Mechanistically, GSDMB N-terminal domain interacts with the key components of the autophagy machinery LC3B and Rab7, facilitating the Rab7 activation during pro-survival autophagy in response to anti-HER2 therapies. Finally, we validated these results in clinical samples where GSDMB/Rab7/LC3B co-expression associates significantly with relapse in HER2 breast and gastric cancers. Conclusion Our findings uncover for the first time a functional link between GSDMB over-expression and protective autophagy in response to HER2-targeted therapies. GSDMB behaves like an autophagy adaptor and plays a pivotal role in modulating autophagosome maturation through Rab7 activation. Finally, our results provide a new and accessible therapeutic approach for HER2/GSDMB + cancers with adverse clinical outcome.

The induction of pluripotency by enforced expression of different sets of genes in somatic cells has been achieved with reprogramming technologies first described by Yamanaka’s group. Methodologies for generating induced pluripotent stem cells are as varied as the combinations of genes used. It has previously been reported that the adenoviral E1a gene can induce the expression of two of the Yamanaka factors (c-Myc and Oct-4) and epigenetic changes. Here, we demonstrate that the E1a-12S over-expression is sufficient to induce pluripotent-like characteristics closely to epiblast stem cells in mouse embryonic fibroblasts through the activation of the pluripotency gene regulatory network. These findings provide not only empirical evidence that the expression of one single factor is sufficient for partial reprogramming but also a potential mechanistic explanation for how viral infection could lead to neoplasia if they are surrounded by the appropriate environment or the right medium, as happens with the tumorogenic niche.

Background and purpose: Ovarian cancer (OC) is the deadliest gynaecologic cancer characterised by a high heterogeneity not only at the clinical point of view but also at the molecular level. This review focuses on the new insights about the OC molecular classification. Materials and methods: We performed a bibliographic search for different indexed articles focused on the new molecular classification of OC. All of them have been published in PubMed and included information about the most frequent molecular alterations in OC confirmed by omics approaches. In addition, we have extracted information about the role of liquid biopsy in the OC diagnosis and prognosis. Results: New molecular insights into OC have allowed novel clinical entities to be defined. Among OC, high-grade serous ovarian carcinoma (HGSOC) which is the most common OC is characterised by omics approaches, mutations in TP53 and in other genes involved in the homologous recombination repair, especially BRCA1/2. Recent studies in HGSOC have allowed a new molecular classification in subgroups according to their mutational, transcriptional, methylation and copy number variation signatures with a real impact in the characterisation of new therapeutic targets for OC to be defined. Furthermore, despite the intrinsic intra-tumour heterogeneity, the advances in next generation sequencing (NGS) analyses of ascetic liquid from OC have opened new ways for its characterisation and treatment. Conclusions: The advances in genomic approaches have been used for the identification of new molecular profiling techniques which define OC subgroups and has supposed advances in the diagnosis and in the personalised treatment of OC.

Gasdermins (GSDM) genes play complex roles in inflammatory diseases and cancer. Gasdermin-B (GSDMB) is frequently upregulated in human cancers, especially in HER2-amplified breast carcinomas, and can promote diverse pro-tumor functions (invasion, metastasis, therapy-resistance). In particular, the GSDMB shortest translated variant (isoform 2; GSDMB2) increases aggressive behavior in breast cancer cells. Paradoxically, GSDMB can also have tumor suppressor (cell death induction) effects in specific biological contexts. However, whether GSDMB has inherent oncogenic, or tumor suppressor function in vivo has not been demonstrated yet in preclinical mouse models, since mice lack GSDMB orthologue. Therefore, to decipher GSDMB cancer functions in vivo we first generated a novel knock-in mouse model (R26-GB2) ubiquitously expressing human GSDMB2. The comprehensive histopathological analysis of multiple tissues from 75 animals showed that nucleus-cytoplasmic GSDMB2 expression did not clearly affect the overall frequency nor the histology of spontaneous neoplasias (mostly lung carcinomas), but associated with reduced incidence of gastric tumors, compared to wildtype animals. Next, to assess specifically the GSDMB2 roles in breast cancer, we generated two additional double transgenic mouse models, that co-express GSDMB2 with either the HER2/NEU oncogene (R26-GB2/MMTV-NEU mice) or the Polyoma middle-T antigen (R26-GB2/MMTV-PyMT) in breast tumors. Consistent with the pro-tumor effect of GSDMB in HER2+ human breast carcinomas, R26-GB2/MMTV-NEU GSDMB2-positive mice have double breast cancer incidence than wildtype animals. By contrast, in the R26-GB2/MMTV-PyMT model of fast growing and highly metastatic mammary tumors, GSDMB2 expression did not significantly influence cancer development nor metastatic potential. In conclusion, our data prove that GSDMB2 in vivo pro-tumor effect is evidenced only in specific biological contexts (in concert with the HER2 oncogene), while GSDMB2 alone does not have overall intrinsic oncogenic potential in genetically modified mice. Our novel models are useful to identify the precise stimuli and molecular mechanisms governing GSDMB functions in neoplasias and can be the basis for the future development of additional tissue-specific and context-dependent cancer models.

Adipose tissue secretes various peptides, including leptin. This hormone acts through the leptin receptor (Ob-R), which is expressed ubiquitously on the surface of various cells, including breast cancer cells and immune cells. Increasing evidence points to an interaction between the tumor microenvironment, tumor cells, and the immune system. Leptin plays an important role in breast cancer tumorigenesis and may be implicated in activation of the immune system. While breast cancer cannot be considered an immunogenic cancer, the triple-negative subtype is an exception. Specific immune cells - tumor infiltrating lymphocytes - are involved in the immune response and act as predictive and prognostic factors in certain breast cancer subtypes. The aim of this article is to review the interaction between adipose tissue, through the expression of leptin and its receptor, and the adaptive immune system in breast cancer.

Breast cancer is the most common tumor in women worldwide, and an increasing public health concern. Knowledge of both protective and negative risk factors is essential for a better understanding of this heterogenous disease. We undertook a review of the recent literature and evaluated the relationship between obesity mediators and breast cancer development depending on menopausal status. Excess weight is now pandemic and has replaced tobacco as the main lifestyle-related risk factor for premature death. Although the prevalence of obesity/overweight has increased globally over the last 50 years, the potential harm attributable to excess fat has generally been underestimated. The relationship between overweight/obesity, breast cancer and overall risk appears to be highly dependent on menopausal status. Thus, obesity increases the risk of breast cancer in postmenopausal women but, conversely, it appears to be protective in premenopausal women. We evaluate the role of different clinical factors potentially involved in this seemingly contradictory relationship, including estrogen, mammogram density, adipokines, insulin-signaling pathway activation, and inflammatory status. A key focus of this review is to better understand the impact of body mass index and menopausal status on these clinical factors and, hence, provide some clarity into the inter-relationships involved in this controversial issue.

The role of prostaglandin (PG) F2α has been scarcely studied in cancer. We have identified a new function for PGF2α in ovarian cancer, stimulating the production of Prostate Transmembrane Protein, Androgen Induced 1 (PMEPA1). We show that this induction increases cell plasticity and proliferation, enhancing tumor growth through PMEPA1. Thus, PMEPA1 overexpression in ovarian carcinoma cells, significantly increased cell proliferation rates, whereas PMEPA1 silencing decreased proliferation. In addition, PMEPA1 overexpression buffered TGFβ signaling, via reduction of SMAD-dependent signaling. PMEPA1 overexpressing cells acquired an epithelial morphology, associated with higher E-cadherin expression levels while β-catenin nuclear translocation was inhibited. Notwithstanding, high PMEPA1 levels also correlated with epithelial to mesenchymal transition markers, such as vimentin and ZEB1, allowing the cells to take advantage of both epithelial and mesenchymal characteristics, gaining in cell plasticity and adaptability. Interestingly, in mouse xenografts, PMEPA1 overexpressing ovarian cells had a clear survival and proliferative advantage, resulting in higher metastatic capacity, while PMEPA1 silencing had the opposite effect. Furthermore, high PMEPA1 expression in a cohort of advanced ovarian cancer patients was observed, correlating with E-cadherin expression. Most importantly, high PMEPA1 mRNA levels were associated with lower patient survival.

Abstract Background Lack of effective tumor-specific delivery systems remains an unmet clinical challenge for successful translation of innovative therapies, such as, therapeutic oligonucleotides. In the past decade, exosomes have been suggested to be ideal drug delivery systems with application in a broad range of pathologies including cancer, due to their organotropic properties. Tumor-derived exosomes, having tumor-homing properties, can efficiently reach cancer cells and therefore behave as carriers for improved drug delivery to the primary tumor and metastases. However, due to their complex composition, and still undefined biological functions, safety concerns arise hampering their translation to the clinics. Results We propose here the development of exosome-mimetic nanosystems (EMNs) that simulate natural tumor-derived exosomes with respect to their structure and functionality, but with a controlled composition, for the targeted delivery of therapeutic oligonucleotides to lung adenocarcinoma cells (microRNA-145 mimics). Making use of the well-known liposome technology, EMNs can be engineered, loaded with the therapeutic compounds, and tailored with specific proteins (integrin α6β4) providing them organotropic properties. EMNs show great similarities to natural exosomes with respect to their physicochemical properties, drug loading capacity, and ability to interact with the cancer target cells in vitro and in vivo, but are easier to manufacture, can be produced at high yields, and are safer by definition. Conclusions We have designed a multifunctional nanoplatform mimicking exosomes, EMNs, and proved their potential to reach cancer cells with a similar efficient that tumor-derived exosomes but providing important advantages in terms of production methodology and regulations. Additionally, EMNs are highly versatile systems that can be tunable for a broader range of applications.

Abstract Background Breast cancer is the second most common cause of cancer-related death among women. Advances in our understanding of the disease have translated into better diagnostics and more effective therapeutics, leading to earlier detection and improved outcomes. Several studies have pointed at lifestyle and environmental factors as contributory for the onset and progression of the disease. Obesity and cholesterol stand out for their potential causal relationship with breast cancer and ease of modification. Main text Obesity and cholesterol represent risk factors for breast cancer, but their impact is largely affected by cofounding variables including menopausal status, disease subtype, and inflammation. Establishing a causal relationship between lifestyle factors and clinical outcomes may be challenging. Epidemiological studies and meta-analyses have rendered conflicting or sometimes contradictory results, possibly owing to the multifactorial nature of the disease. We discuss the supporting evidence and limitations in our understanding of obesity and cholesterol as risk factors for breast cancer. Conclusions There is sufficient evidence that obesity and cholesterol impact clinical outcomes. Physicians are advised to take steps to help patients with their weight, such as recommending dietary and lifestyle interventions.

Myeloid-derived suppressor cells (MDSCs) and dendritic cells (DCs) arise from common progenitors. Tumor-derived factors redirect differentiation from immune-promoting DCs to tolerogenic MDSCs, an immunological hallmark of cancer. Indeed, in vitro differentiation of DCs from human primary monocytes results in the generation of MDSCs under tumor-associated conditions (PGE2 or tumor cell-conditioned media). Comparison of MDSC and DC DNA methylomes now reveals extensive demethylation with specific gains of DNA methylation and repression of immunogenic-associated genes occurring in MDSCs specifically, concomitant with increased DNA methyltransferase 3A (DNMT3A) levels. DNMT3A downregulation erases MDSC-specific hypermethylation, and it abolishes their immunosuppressive capacity. Primary MDSCs isolated from ovarian cancer patients display a similar hypermethylation signature in connection with PGE2-dependent DNMT3A overexpression. Our study links PGE2- and DNMT3A-dependent hypermethylation with immunosuppressive MDSC functions, providing a promising target for therapeutic intervention.

Current screening methods for ovarian cancer can only detect advanced disease. Earlier detection has proved difficult because the molecular precursors involved in the natural history of the disease are unknown. To identify early driver mutations in ovarian cancer cells, we used dense whole genome sequencing of micrometastases and microscopic residual disease collected at three time points over three years from a single patient during treatment for high-grade serous ovarian cancer (HGSOC). The functional and clinical significance of the identified mutations was examined using a combination of population-based whole genome sequencing, targeted deep sequencing, multi-center analysis of protein expression, loss of function experiments in an in-vivo reporter assay and mammalian models, and gain of function experiments in primary cultured fallopian tube epithelial (FTE) cells. We identified frequent mutations involving a 40 kb distal repressor region for the key stem cell differentiation gene SOX2. In the apparently normal FTE, the region was also mutated. This was associated with a profound increase in SOX2 expression (p

Background: Recent studies showed a relevant role of hematogenous spread in ovarian cancer and the interest of circulating tumor cells (CTCs) monitoring as a prognosis marker. The aim of the present study was the characterization of CTCs from ovarian cancer patients, paying special attention to cell plasticity characteristics to better understand the biology of these cells. Methods: CTCs isolation was carried out in 38 patients with advanced high-grade serous ovarian cancer using in parallel CellSearch and an alternative EpCAM-based immunoisolation followed by RT-qPCR analysis to characterize these cells. Results: Epithelial CTCs were found in 21% of patients, being their presence higher in patients with extraperitoneal metastasis. Importantly, this population was characterized by the expression of epithelial markers as MUC1 and CK19, but also by genes associated with mesenchymal and more malignant features as TIMP1, CXCR4 and the stem markers CD24 and CD44. In addition, we evidenced the relevance of TIMP1 expression to promote tumor proliferation, suggesting its interest as a therapeutic target. Conclusions: Overall, we evidenced the utility of the molecular characterization of EpCAM+ CTCs from advanced ovarian cancer patients to identify biomarkers with potential applicability for disseminated disease detection and as therapeutic targets such as TIMP1.

Summary Lysyl-oxidase-like 2 (LOXL2) is a member of the lysyl oxidase family that catalyzes the cross-linking of collagens or elastins in the extracellular matrix, thus regulating the tensile strength of tissues. However, many reports have suggested different intracellular roles for LOXL2, including the ability to regulate gene transcription and tumor progression. We previously reported that LOXL2 mediates epithelial-to-mesenchymal transition (EMT) by Snail1-dependent and independent mechanisms, related to E-cadherin silencing and downregulation of epidermal differentiation and cell polarity components, respectively. Whether or not the catalytic activity of LOXL2 is required to induce/sustain EMT is actually unknown. Here we show that LOXL2 catalytic inactive mutants collaborate with Snail1 in E-cadherin gene repression to trigger EMT and, in addition, promote FAK/Src pathway activation to support EMT. These findings reveal a non-conventional role of LOXL2 on regulating epithelial cell plasticity.

Gasdermin B (GSDMB) belongs to the Gasdermin protein family that comprises four members (GSDMA-D). Gasdermin B expression has been detected in some tumor types such as hepatocarcinomas, gastric and cervix cancers; and its over-expression has been related to tumor progression. At least four splicing isoforms of GSDMB have been identified, which may play differential roles in cancer. However, the implication of GSDMB in carcinogenesis and tumor progression is not well understood. Here, we uncover for the first time the functional implication of GSDMB in breast cancer. Our data shows that high levels of GSDMB expression is correlated with reduced survival and increased metastasis in breast cancer patients included in an expression dataset (>1,000 cases). We demonstrate that GSDMB is upregulated in breast carcinomas compared to normal breast tissue, being the isoform 2 (GSDMB-2) the most differentially expressed. In order to evaluate the functional role of GSDMB in breast cancer two GSDMB isoforms were studied (GSDMB-1 and GSDMB-2). The overexpression of both isoforms in the MCF7 breast carcinoma cell line promotes cell motility and invasion, while its silencing in HCC1954 breast carcinoma cells decreases the migratory and invasive phenotype. Importantly, we demonstrate that both isoforms have a differential role on the activation of Rac-1 and Cdc-42 Rho-GTPases. Moreover, our data support that GSMDB-2 induces a pro-tumorigenic and pro-metastatic behavior in mouse xenograft models as compared to GSDMB-1. Finally, we observed that although both GSDMB isoforms interact in vitro with the chaperone Hsp90, only the GSDMB-2 isoform relies on this chaperone for its stability. Taken together, our results provide for the first time evidences that GSDMB-2 induces invasion, tumor progression and metastasis in MCF7 cells and that GSDMB can be considered as a new potential prognostic marker in breast cancer.

Squamous cell lung cancer (SCC) and adenocarcinoma are the most common histological subtypes of non-small cell lung cancer (NSCLC), and have been traditionally managed in the clinic as a single entity. Increasing evidence, however, illustrates the biological diversity of these two histological subgroups of lung cancer, and supports the need to improve our understanding of the molecular basis beyond the different phenotypes if we aim to develop more specific and individualized targeted therapy. The purpose of this study was to identify microRNA (miRNA)-dependent transcriptional regulation differences between SCC and adenocarcinoma histological lung cancer subtypes. In this work, paired miRNA (667 miRNAs by TaqMan Low Density Arrays (TLDA)) and mRNA profiling (Whole Genome 44 K array G112A, Agilent) was performed in tumor samples of 44 NSCLC patients. Nine miRNAs and 56 mRNAs were found to be differentially expressed in SCC versus adenocarcinoma samples. Eleven of these 56 mRNA were predicted as targets of the miRNAs identified to be differently expressed in these two histological conditions. Of them, 6 miRNAs (miR-149, miR-205, miR-375, miR-378, miR-422a and miR-708) and 9 target genes (CEACAM6, CGN, CLDN3, ABCC3, MLPH, ACSL5, TMEM45B, MUC1) were validated by quantitative PCR in an independent cohort of 41 lung cancer patients. Furthermore, the inverse correlation between mRNAs and microRNAs expression was also validated. These results suggest miRNA-dependent transcriptional regulation differences play an important role in determining key hallmarks of NSCLC, and may provide new biomarkers for personalized treatment strategies.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

The human ghrelin gene, which encodes the ghrelin and obestatin peptides, contains 5 exons (Ex), with Ex1-Ex4 encoding a 117 amino-acid (aa) preproprotein that is known to be processed to yield a 28-aa (ghrelin) and/or a 23-aa (obestatin) mature peptides, which possess biological activities in multiple tissues. However, the ghrelin gene also encodes additional peptides through alternative splicing or post-translational modifications. Indeed, we previously identified a spliced mRNA ghrelin variant in mouse (In2-ghrelin-variant), which is regulated in a tissue-dependent manner by metabolic status and may thus be of biological relevance. Here, we have characterized a new human ghrelin variant that contains Ex0-1, intron (In) 1, and Ex2 and lacks Ex3-4. This human In1-ghrelin variant would encode a new prepropeptide that conserves the first 12aa of native-ghrelin (including the Ser3-potential octanoylation site) but has a different C-terminal tail. Expression of In1-variant was detected in 22 human tissues and its levels were positively correlated with those of ghrelin-O-acyltransferase (GOAT; p = 0.0001) but not with native-ghrelin expression, suggesting that In1-ghrelin could be a primary substrate for GOAT in human tissues. Interestingly, levels of In1-ghrelin variant expression in breast cancer samples were 8-times higher than those of normal mammary tissue, and showed a strong correlation in breast tumors with GOAT (p = 0.0001), ghrelin receptor-type 1b (GHSR1b; p = 0.049) and cyclin-D3 (a cell-cycle inducer/proliferation marker; p = 0.009), but not with native-ghrelin or GHSR1a expression. Interestingly, In1-ghrelin variant overexpression increased basal proliferation of MDA-MB-231 breast cancer cells. Taken together, our results provide evidence that In1-ghrelin is a novel element of the ghrelin family with a potential pathophysiological role in breast cancer.