Acquisition Of Stem Cell Properties Research Articles

Abstract 5451: FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer

Abstract Cancer stem cells (CSCs) pose a significant clinical challenge due to their inherent resistance to therapies and prevalence in cancer recurrence. CSCs have the ability for self-renewal and to adapt to different microenvironments during dissemination and seeding into distal organs, and these properties are critical in metastasis. We and others have shown that epithelial to mesenchymal transition (EMT) drives the acquisition of stem cell properties, particularly via the forkhead box protein C2 (FOXC2) transcription factor. FOXC2 is a master regulator of EMT and is necessary for the induction and maintenance of CSC properties. Importantly, FOXC2 is not expressed in most adult tissues except adipocytes, in which it dictates several aspects of lipid metabolism. Additionally, metabolic changes during EMT can equip cancer cells with enhanced survival mechanisms during tumor progression and metastasis. We hypothesized that FOXC2 regulates metabolism in EMT-induced cells, facilitating stem cell properties and metastatic competence. To determine the downstream metabolic genes regulated by FOXC2 and that enhance stemness properties in cancer cells, we performed gene expression profiling and identified fatty acid binding protein 4 (FABP4), which is known to be involved in lipid metabolism, especially in adipocytes and macrophages. We demonstrate that inhibition of FOXC2 expression using shRNA in EMT-induced cells markedly reduces FABP4 expression. In agreement with this, we found that the overexpression of FOXC2 expression rescues the expression of FABP4. In addition, FOXC2 binds to the FABP4 promoter and regulates its expression suggesting that FABP4 is a direct target of FOXC2. We demonstrate that both lipid uptake and CSC formation is inhibited in a dose-dependent manner using a FABP4-specific small molecule inhibitor. Additional experiments are underway to assess the role of FABP4 in CSC formation in vivo and in contributions to metastasis, using our small molecule inhibitor in mouse models of metastatic breast cancer. Overall, our data indicate that FABP4 is a key regulator of stemness in breast cancer cells and targeting the FOXC2—FABP4 axis may provide therapeutic benefits for breast cancer. This work also provides insights into the metabolic shifts that occur during EMT and CSC formation, which warrants further investigation into these mechanisms. Citation Format: Breanna R. Demestichas, Vinita Shivakumar, Esmeralda Ramirez-Peña, Joseph Taube, Petra den Hollander, Sendurai A. Mani. FOXC2 influences EMT-induced stem cell properties through regulation of FABP4 in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5451.

NDRG2 regulates the formation of reactive astrocyte-derived progenitor cells via Notch signaling pathway after brain traumatic injury in rats.

In response to traumatic brain injury, a subpopulation of cortical astrocytes is activated, resulting in acquisition of stem cell properties, known as reactive astrocytes-derived progenitor cells (Rad-PCs). However, the underlying mechanisms remain largely unknown during this process. In this study, we examined the role of N-myc downstream-regulated gene 2 (NDRG2), a differentiation- and stress-associated molecule, in Rad-PCs after cortical stab injury in adult rats. Immunohistochemical analysis showed that in the cerebral cortex of normal adult rats, NDRG2 was exclusively expressed in astrocytes. After liu cortical injury, the expression of NDRG2 was significantly elevated around the wound and most cells expressing NDRG2 also expressed GFAP, a reactive astrocyte marker. Importantly, NDRG2-expressing cells were co-labeled with Nestin, a marker for neural stem cells, some of which also expressed cell proliferation marker Ki67. Overexpression of NDRG2 further increased the number of NDRG2/Nestin double-labeling cells around the lesion. In contrast, shRNA knockdown of NDRG2 decreased the number of NDRG2+/Nestin+ cells. Intracerebroventricular administration of stab-injured rats with a Notch antagonist, DAPT, led to a significant decrease in Nestin+/NDRG2+ cells around the injured boundary, but did not affect NDRG2+ cells. Moreover, overexpression or knockdown of NDRG2 led to up- and down-regulation of the expression of Notch intracellular domain NICD and Notch target gene Hes1, respectively. Taken together, these results suggest that NDRG2 may play a role in controlling the formation of Rad-PCs in the cerebral cortex of adult rats following traumatic injury, and that Notch signaling pathway plays a key role in this process.

Epithelial-to-Mesenchymal Transition in Metastasis: Focus on Laryngeal Carcinoma.

In epithelial neoplasms, such as laryngeal carcinoma, the survival indexes deteriorate abruptly when the tumor becomes metastatic. A molecular phenomenon that normally appears during embryogenesis, epithelial-to-mesenchymal transition (EMT), is reactivated at the initial stage of metastasis when tumor cells invade the adjacent stroma. The hallmarks of this phenomenon are the abolishment of the epithelial and acquisition of mesenchymal traits by tumor cells which enhance their migratory capacity. EMT signaling is mediated by complex molecular pathways that regulate the expression of crucial molecules contributing to the tumor’s metastatic potential. Effectors of EMT include loss of adhesion, cytoskeleton remodeling, evasion of apoptosis and immune surveillance, upregulation of metalloproteinases, neovascularization, acquisition of stem-cell properties, and the activation of tumor stroma. However, the current approach to EMT involves a holistic model that incorporates the acquisition of potentials beyond mesenchymal transition. As EMT is inevitably associated with a reverse mesenchymal-to-epithelial transition (MET), a model of partial EMT is currently accepted, signifying the cell plasticity associated with invasion and metastasis. In this review, we identify the cumulative evidence which suggests that various aspects of EMT theory apply to laryngeal carcinoma, a tumor of significant morbidity and mortality, introducing novel molecular targets with prognostic and therapeutic potential.

Role of EMT in the DNA damage response, double-strand break repair pathway choice and its implications in cancer treatment.

Numerous epithelial–mesenchymal transition (EMT) characteristics have now been demonstrated to participate in tumor development. Indeed, EMT is involved in invasion, acquisition of stem cell properties, and therapy‐associated resistance of cancer cells. Together, these mechanisms offer advantages in adapting to changes in the tumor microenvironment. However, recent findings have shown that EMT‐associated transcription factors (EMT‐TFs) may also be involved in DNA repair. A better understanding of the coordination between the DNA repair pathways and the role played by some EMT‐TFs in the DNA damage response (DDR) should pave the way for new treatments targeting tumor‐specific molecular vulnerabilities, which result in selective destruction of cancer cells. Here we review recent advances, providing novel insights into the role of EMT in the DDR and repair pathways, with a particular focus on the influence of EMT on cellular sensitivity to damage, as well as the implications of these relationships for improving the efficacy of cancer treatments.

Recent Advances in Developmental Hematopoiesis: Diving Deeper With New Technologies.

The journey of a hematopoietic stem cell (HSC) involves the passage through successive anatomical sites where HSCs are in direct contact with their surrounding microenvironment, also known as niche. These spatial and temporal cellular interactions throughout development are required for the acquisition of stem cell properties, and for maintaining the HSC pool through balancing self-renewal, quiescence and lineage commitment. Understanding the context and consequences of these interactions will be imperative for our understanding of HSC biology and will lead to the improvement of in vitro production of HSCs for clinical purposes. The aorta-gonad-mesonephros (AGM) region is in this light of particular interest since this is the cradle of HSC emergence during the embryonic development of all vertebrate species. In this review, we will focus on the developmental origin of HSCs and will discuss the novel technological approaches and recent progress made to identify the cellular composition of the HSC supportive niche and the underlying molecular events occurring in the AGM region.

Translocation of intracellular CD24 constitutes a triggering event for drug resistance in breast cancer

The capacity of tumor cells to shift dynamically between different states could be responsible for chemoresistance and has been commonly linked to the acquisition of stem cell properties. Here, we have evaluated the phenotype switching associated with drug resistance in breast cancer cell lines and cell lineage obtained from Brazilian patients. We have highlighted the role of the cancer stem cell marker CD24 in the dynamics of cell plasticity and the acquirement of drug resistance. We showed that the translocation of CD24 from cytosol to cell membrane is a triggering event for the phenotype change of breast tumor cells exposed to drug stress. Here, we provide evidence that the phenotype switching is due to the presence of a cytosolic pool of CD24. Importantly, the cellular localization of CD24 was correlated with the changes in the dynamics of p38 MAPK activation. A strong and continuous phosphorylation of the p38 MAPK led to the overexpression of Bcl-2 after treatment in persistent cells presenting high density of CD24 on cell membrane. This phenotype enabled the cells to enter in slow-down of cell cycle, after which several weeks later, the dormant cells proliferated again. Importantly, the use of a p38 activity inhibitor sensitized cells to drug treatment and avoided chemoresistance.

Nicotinamide N-Methyltransferase in Acquisition of Stem Cell Properties and Therapy Resistance in Cancer.

The activity of nicotinamide N-methyltransferase (NNMT) is tightly linked to the maintenance of the nicotinamide adenine dinucleotide (NAD+) level. This enzyme catalyzes methylation of nicotinamide (NAM) into methyl nicotinamide (MNAM), which is either excreted or further metabolized to N1-methyl-2-pyridone-5-carboxamide (2-PY) and H2O2. Enzymatic activity of NNMT is important for the prevention of NAM-mediated inhibition of NAD+-consuming enzymes poly–adenosine -diphosphate (ADP), ribose polymerases (PARPs), and sirtuins (SIRTs). Inappropriately high expression and activity of NNMT, commonly present in various types of cancer, has the potential to disrupt NAD+ homeostasis and cellular methylation potential. Largely overlooked, in the context of cancer, is the inhibitory effect of 2-PY on PARP-1 activity, which abrogates NNMT’s positive effect on cellular NAD+ flux by stalling liberation of NAM and reducing NAD+ synthesis in the salvage pathway. This review describes, and discusses, the mechanisms by which NNMT promotes NAD+ depletion and epigenetic reprogramming, leading to the development of metabolic plasticity, evasion of a major tumor suppressive process of cellular senescence, and acquisition of stem cell properties. All these phenomena are related to therapy resistance and worse clinical outcomes.

Interplay between Metabolism Reprogramming and Epithelial-to-Mesenchymal Transition in Cancer Stem Cells.

Simple SummaryTumor cells display important plasticity potential. Notably, tumor cells have the ability to change toward immature cells called cancer stem cells under the influence of the tumor environment. Importantly, cancer stem cells are a small subset of relatively quiescent cells that, unlike rapidly dividing differentiated tumor cells, escape standard chemotherapies, causing relapse or recurrence of cancer. Interestingly, these cells adopt a specific metabolism. Most often, they mainly rely on glucose uptake and metabolism to sustain their energy needs. This metabolic reprogramming is set off by environmental factors such as pro-inflammatory signals or catecholamine hormones (epinephrine, norepinephrine). A better understanding of this process could provide opportunities to kill cancer stem cells. Indeed, it would become possible to develop drugs that act specifically on metabolic pathways used by these cells. These new drugs could be used to strengthen the effects of current chemotherapies and overcome cancers with poor prognoses.Tumor cells display important plasticity potential, which contributes to intratumoral heterogeneity. Notably, tumor cells have the ability to retrodifferentiate toward immature states under the influence of their microenvironment. Importantly, this phenotypical conversion is paralleled by a metabolic rewiring, and according to the metabostemness theory, metabolic reprogramming represents the first step of epithelial-to-mesenchymal transition (EMT) and acquisition of stemness features. Most cancer stem cells (CSC) adopt a glycolytic phenotype even though cells retain functional mitochondria. Such adaptation is suggested to reduce the production of reactive oxygen species (ROS), protecting CSC from detrimental effects of ROS. CSC may also rely on glutaminolysis or fatty acid metabolism to sustain their energy needs. Besides pro-inflammatory cytokines that are well-known to initiate the retrodifferentiation process, the release of catecholamines in the microenvironment of the tumor can modulate both EMT and metabolic changes in cancer cells through the activation of EMT transcription factors (ZEB1, Snail, or Slug (SNAI2)). Importantly, the acquisition of stem cell properties favors the resistance to standard care chemotherapies. Hence, a better understanding of this process could pave the way for the development of therapies targeting CSC metabolism, providing new strategies to eradicate the whole tumor mass in cancers with unmet needs.

KHDRBS3 promotes multi-drug resistance and anchorage-independent growth in colorectal cancer.

5‐Fluorouracil (5‐FU) is one of the most frequently used pharmacological agents in the treatment of colorectal cancer (CRC). Resistance to chemotherapy is a major cause of treatment failure of CRC, and it is a well known fact that cancer stem cells play a significant role in the acquisition of drug resistance. In this study, we focused on the KHDRBS3 gene that encodes KH RNA Binding Domain Containing, Signal Transduction Associated 3. We first clarified the relationship between KHDRBS3 and 5‐FU resistance. We then observed higher expression levels of KHDRBS3 in KRAS‐mutant organoids and cell lines in comparison with KRAS wild‐type organoids and cell lines. Immunohistochemical analysis using CRC cases revealed that the prognosis of KHDRBS3‐positive patients was significantly worse compared with that of KHDRBS3‐negative patients. Univariate and multivariate Cox proportional hazards analyses showed that KHDRBS3 was an independent prognostic factor in patients with CRC. We determined that KHDRBS3 might play a crucial role in the acquisition of stem cell properties, such as drug resistance and spheroid/organoid formation, by regulating CD44 variant expression and the Wnt signaling pathway. In an immunodeficient mouse model, KHDRBS3‐positive cells showed efficient tumor formation and formed metastatic lesions in the lungs. These results indicated that KHDRBS3 plays a crucial role in drug resistance and anchorage‐independent growth by maintaining stem cell‐like features in CRC cells. KHDRBS3 could be a promising candidate marker for predicting chemotherapeutic effect and prognosis in CRC patients.

The role of TRIM family proteins in the regulation of cancer stem cell self-renewal.

The tripartite‐motif (TRIM) family of proteins represents one of the largest classes of putative single protein RING‐finger E3 ubiquitin ligases. The members of this family are characterized by an N‐terminal TRIM motif containing one RING‐finger domain, one or two zinc‐finger domains called B boxes (B1 box and B2 box), and a coiled‐coil region. The TRIM motif can be found in isolation or in combination with a variety of C‐terminal domains, and based on C‐terminus, TRIM proteins are classified into 11 distinct groups. Because of the complex nature of TRIM proteins, they are implicated in a variety of cellular functions and biological processes, including regulation of cell proliferation, cell division and developmental processes, cancer transformation, regulation of cell metabolism, autophagocytosis, modification of chromatin status, regulation of gene transcription, post‐translational modifications, and interactions with pathogens. Here, we demonstrate the specific activities of TRIM family proteins that contribute to the cancer stem cell phenotype. A growing body of evidence demonstrates that several TRIM members guarantee the acquisition of stem cell properties and the ability to sustain stem‐like phenotype by cancer cells using distinct mechanisms. For other members, further work is needed to understand their full contribution to stem cell self‐renewal. Identification of TRIM proteins that possess the potential to serve as therapeutic targets may result in the development of new therapeutic strategies. Finally, these strategies may result in the disruption of the machinery of stemness acquisition, which may prevent tumor growth, progression, and overcome the resistance to anticancer therapies.

Wounding induces dedifferentiation of epidermal Gata6+ cells and acquisition of stem cell properties.

The epidermis is maintained by multiple stem cell populations whose progeny differentiate along diverse, and spatially distinct, lineages. Here we show that the transcription factor Gata6 controls the identity of the previously uncharacterized sebaceous duct (SD) lineage and identify the Gata6 downstream transcription factor network that specifies a lineage switch between sebocytes and SD cells. During wound healing differentiated Gata6+ cells migrate from the SD into the interfollicular epidermis and dedifferentiate, acquiring the ability to undergo long-term self-renewal and differentiate into a much wider range of epidermal lineages than in undamaged tissue. Our data not only demonstrate that the structural and functional complexity of the junctional zone is regulated by Gata6, but also reveal that dedifferentiation is a previously unrecognized property of post-mitotic, terminally differentiated cells that have lost contact with the basement membrane. This resolves the long-standing debate about the contribution of terminally differentiated cells to epidermal wound repair.

Senescence-associated IL-6 and IL-8 cytokines induce a self- and cross-reinforced senescence/inflammatory milieu strengthening tumorigenic capabilities in the MCF-7 breast cancer cell line

BackgroundThere is compelling evidence associating senescent cells with the malignant progression of tumours. Of all senescence-related mechanisms, the so-called senescence-associated secretory phenotype (SASP) has attracted much attention. Since the pro-inflammatory cytokines IL-6 and IL-8 are consistently present in the SASP, and secreted by highly aggressive breast cancer cell lines, we aimed at elucidating their role on the less aggressive breast cancer cell line MCF-7, which does not secret these cytokines.MethodsThe MCF-7 cell line was treated with either senescence-conditioned medium (SCM), IL-6 or IL-8 and then evaluated for phenotypic (CD44 and CD24 by FACS) and functional changes associated with an EMT program (migration/invasion) and for the acquisition of stem cell properties: mammosphere-forming capacity, expression of reprogramming factors (by qRT-PCR) and multilineage differentiation potential. We also evaluated the role of IL6 and IL8 in the cytokine-secreting, highly tumorigenic cell line MDA-MB-231.ResultsOur results show that treatment of MCF-7 cells with IL6 and IL8, alone or together, induced the appearance of cells with fibroblastoid morphology, increased CD44 expression and migration, self-renewal and multilineage differentiation capacity, all characteristics compatible with an EMT program and stemness. These changes closely resembled those induced by a SCM. Interestingly, SCM treatments further increased IL6 and IL8 secretion by MCF-7 cells, thus suggesting the participation of an autocrine loop. Indeed, neutralizing antibodies against IL6 and IL8 reversed the effects of SCM on MCF-7, pinpointing these cytokines as major mediators of EMT and stemness-related effects associated with the senescent microenvironment. Additionally, prolonged exposure of MCF cells to IL6 or IL8 induced the appearance of senescent cells, suggesting a mechanism by which senescence and inflammation are reinforced favouring the acquisition of EMT and stem-like features at the population level, thus increasing tumour aggressiveness. Strikingly, our results also show that both IL6 and IL8 are important to maintain aggressive traits in MDA-MB-231 cells, a highly tumorigenic cell line, which appears to be devoid of stemness-related features.ConclusionsThis study demonstrates that, similar to what is observed with a senescent microenvironment, purified IL6 and IL8 induce a self- and cross-reinforced senescence/inflammatory milieu responsible for the emergence of epithelial plasticity and stemness features, thus conferring more aggressive phenotypes to a luminal breast cancer cell line. On the other hand, the basal-like MDA-MB-231 cells, whose aggressiveness-related features depend on IL6 and IL8 secretion, almost completely lack mammosphere formation and differentiation capacities, suggesting that tumour aggressiveness is not always related to stemness.

Generation and characterization of mice for conditional inactivation of Zeb1.

The multizinc finger containing transcription factor ZEB1 plays crucial roles during various aspects of mammalian development and tumorigenesis. Best studied in human tumors, ZEB1 is activating the embryo-derived program of epithelial-mesenchymal transition (EMT). The aberrant activation of EMT confers an invasive metastasizing phenotype with acquisition of stem cell properties and resistance to radio- and chemotherapy. Although ZEB1 has very important functions in tumor progression, not much is known about its role in physiological contexts and during development and homeostasis. We describe the generation of Zeb1flox/flox mice carrying a targeted mutation for conditional Zeb1 gene inactivation and show that homozygous Zeb1-depletion in the germline results in a phenotype similar to the conventional Zeb1 knockout.

UPA/uPAR and SERPINE1 in head and neck cancer: role in tumor resistance, metastasis, prognosis and therapy

There is strong evidence supporting the role of the plasminogen activator system in head and neck squamous cell carcinoma (HNSCC), particularly of its uPA (urokinase plasminogen activator) / uPAR (urokinase plasminogen activator receptor) and SERPINE1 components. Overexpression of uPA/uPAR and SERPINE1 enhances tumor cell migration and invasion and plays a key role in metastasis development, conferring poor prognosis. The apparent paradox of uPA/uPAR and its inhibitor SERPINE1 producing similar effects is solved by the identification of SERPINE1 activated signaling pathways independent of uPA inhibition. Both uPA/uPAR and SERPINE1 are directly linked to the induction of epithelial-to-mesenchymal transition, the acquisition of stem cell properties and resistance to antitumor agents. The aim of this review is to provide insight on the deregulation of these proteins in all these processes.We also summarize their potential value as prognostic biomarkers or potential drug targets in HNSCC patients. Concomitant overexpression of uPA/uPAR and SERPINE1 is associated with a higher risk of metastasis and could be used to identify patients that would benefit from an adjuvant treatment. In the future, the specific inhibitors of uPA/uPAR and SERPINE1, which are still under development, could be used to design new therapeutic strategies in HNSCCs.

Brachyury as a potential modulator of androgen receptor activity and a key player in therapy resistance in prostate cancer.

Prostate cancer (PCa) is the most commonly diagnosed neoplasm and the second leading cause of cancer-related deaths in men. Acquisition of resistance to conventional therapy is a major problem for PCa patient management. Several mechanisms have been described to promote therapy resistance in PCa, such as androgen receptor (AR) activation, epithelial-to-mesenchymal transition (EMT), acquisition of stem cell properties and neuroendocrine transdifferentiation (NEtD). Recently, we identified Brachyury as a new biomarker of PCa aggressiveness and poor prognosis. In the present study we aimed to assess the role of Brachyury in PCa therapy resistance. We showed that Brachyury overexpression in prostate cancer cells lines increased resistance to docetaxel and cabazitaxel drugs, whereas Brachyury abrogation induced decrease in therapy resistance. Through ChiP-qPCR assays we further demonstrated that Brachyury is a direct regulator of AR expression as well as of the biomarker AMACR and the mesenchymal markers Snail and Fibronectin. Furthermore, in vitro Brachyury was also able to increase EMT and stem properties. By in silico analysis, clinically human Brachyury-positive PCa samples were associated with biomarkers of PCa aggressiveness and therapy resistance, including PTEN loss, and expression of NEtD markers, ERG and Bcl-2. Taken together, our results indicate that Brachyury contributes to tumor chemotherapy resistance, constituting an attractive target for advanced PCa patients.

New Insights into the Crossroads between EMT and Stemness in the Context of Cancer.

The epithelial-mesenchymal transition (EMT) is an example of cellular plasticity, where an epithelial cell acquires a mesenchymal-like phenotype that increases its migratory and invasive properties. Stemness is the ability of stem cells to proliferate in an asymmetric way that allows them to maintain the reservoir of undifferentiated cells with stem cell identity, but also to produce new differentiated cells. Initial works revealed that activation of the EMT program in epithelial cells induces the acquisition of stem cell properties, which in the context of cancer may contribute to the appearance of tumor initiating cells (TIC). However, a number of groups have recently reported that mesenchymal-epithelial transition (MET) is required for efficient metastatic colonization and that EMT may be not necessarily associated with stemness. In this review, we summarize recent findings that extend our knowledge about the crossroads between EMT and stemness and their relevance under physiological or pathological conditions.

Abstract OT1-01-01: A phase I trial of the safety and immunogenicity of a multiple antigen vaccine (STEMVAC) in HER2 negative advanced stage breast cancer patients

Abstract Background: Biologically relevant epithelial to mesenchymal transformation (EMT) associated proteins, a subpopulation of tumor cells with stem like properties, have been identified and are responsible for tumor initiation, metastasis formation and resistance to cancer therapies involved in breast cancer (BC) initiation. It is hypothesized that the acquisition of stem cell properties is driven by EMT induction and that BC stem cells express EMT-associated proteins. A vaccine which would educate the immune system to recognize and eliminate cells that have up-regulated proteins associated with BC stem cells/EMT could eradicate BC at the time of initiation or relapse. We have identified 5 stem cell/EMT proteins that are immunogenic in BC patients and created a vaccine, STEMVAC, composed of extended Th1 epitopes derived from these proteins. STEMVAC is safe and inhibits tumor growth in preclinical murine studies. Trial design: Phase I dose escalation study evaluating 3 doses of STEMVAC admixed with 100 mcg of GMCSF. Patients enrolled sequentially into 1 of 3 dose arms (10 patients/arm): Arm 1=150 mcg, Arm 2=300 mcg, and Arm 3=600 mcg. 3 patients must complete 3 monthly vaccines and month 4 evaluation with no dose limiting toxicity before further accrual to that arm. Patients may receive 2 boosters, 3 and 9 months after their 3rd vaccine. Toxicity is assessed at baseline through end of study. Serial blood draws for immunologic monitoring is done. Eligibility criteria: Stage III-IV HER2 negative BC patients treated with standard therapy who: (1) are without evidence of disease or have stable bone-only disease, (2) are 28 days from last chemotherapy, radiotherapy, systemic steroids, (3) have adequate blood counts, (4) have no active autoimmune disease. Endocrine therapy and bisphosphonates are allowed. Specific aims: (1) Determine the safety of 3 escalating doses of STEMVAC, (2) Determine the most immunogenic dose, (3) Determine whether a STEMVAC Th1 polyepitope plasmid based vaccine elicits persistent T cell memory, and (4) Evaluate if STEMVAC modulates T regulatory and myeloid derived suppressor cells. Statistical methods: Safety will be determined by laboratory and clinical parameters. Descriptive statistics will be used to summarize changes from baseline. Safety benchmarks to move to the next arm will be grade 3 toxicity rate of ≤ 15% and grade 4 toxicity rate of ≤ 5% in the first 3 vaccinations. Immunogenicity evaluated by generation of antigen specific Th1 immunity via ELISPOT. Immunologic efficacy is defined as achievement of significant increase in Th1 immunity for ≥50% of the immunizing antigens compared to baseline; and a greater proportion of patients developing T-cell immunity to a greater number of the antigens included in the vaccine. Exploratory analysis will be used to assess memory Th1 dominant immune response to all 5 antigens. Treg and MDSC will be defined as present or absent, and the probability of each will be estimated as a simple proportion. Target accrual: 30 patients-no patients accrued to date. Citation Format: Higgins DM, Childs JS, Salazar LG, Disis ML. A phase I trial of the safety and immunogenicity of a multiple antigen vaccine (STEMVAC) in HER2 negative advanced stage breast cancer patients. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr OT1-01-01.

Stem Cells in Translational Cancer Research.

Since the seminal studies by Fialkow et al. in the seventies [1] and by Bonnet and Dick in the nineties [2], where an organized tumor cell hierarchy originating from transformed hematopoietic stem cells was found in myeloid leukemias, the cancer stem cell (CSC) model of tumor development drew the attention of the scientific community and became the focus of extensive discussion. This theme gained momentum over the past decade, with the identification of stemlike cells as drivers of tumor initiation, recurrence, and metastasis spread in a variety of nonhematopoietic human cancers. These characteristics postulated them as novel and promising therapeutic targets for cancer, especially in the context of drug-resistant tumors [3]. The origin of these stemlike cells is being extensively studied. In some types of tumors, such as in blood, brain, colon, and skin cancer, it has been shown that adult stem cells are prone to transformation due to a longer half-life within tissues compared to their cell progenies, facilitating the accumulation of oncogenic mutations as a result of prolonged exposure to genotoxic stresses [4]. In other cases, de novo acquisition of stem cell properties may occur in either normal or neoplastic cells due to genetic/epigenetic aberrations [5]. Finally, the CSC model has been put forward to explain why tumors like breast cancer might return years after surgery. In this situation, lower proliferation rate might allow cells to escape from aggressive therapy and allow for genetic/epigenetic evolution. Advances in the field, however, point to varied and more complex models of tumor development than previously envisioned, in which the stemlike phenotype may also be dynamically acquired by cancer cells through interaction with stromal cells and soluble factors [6]. A relevant question timely addressed in this special issue is the understanding of the contribution of resident stem cells to the tumor microenvironment (TME). This is of major relevance since they may significantly influence tumor progression, aggressiveness, and response to therapy. The contribution of pericytes to tumor growth, angiogenesis, metastasis, and evasion of immune destruction, which are classic hallmarks of cancer, is comprehensively reviewed by A. L. Ribeiro and O. K. Okamoto. In their article, the authors also discuss a pericyte-mediated regulation of stemness properties in cancer cells and argue in favor of pericytes as cellular targets for new cancer therapies aiming at the TME. Indeed, many new anticancer therapies targeting the TME are under development, with currently approved antiangiogenic drugs as examples of such strategy. However, events of tumor recurrence and poor response to antiangiogenic therapy still puzzle researchers and emphasize the need for continuous studies. In the article by M. Marcola and C. E. Rodrigues, the involvement of endothelial progenitor cells in tumor angiogenesis is critically reviewed. In their article, the specific roles of different members of the VEGF family of growth factors, as well as the relevance of the vascular niche to stemness in cancer cells, are also carefully discussed. Another practical issue of clinical relevance is the rigorous evaluation of potential oncogenic risks associated with stem cell therapy. Long-term safety issues must be properly addressed before stem cell-based therapies enter clinical trials, but such studies are outnumbered in the literature by studies evaluating therapeutic effects based on restoration of tissue integrity and physiological balance. While the original article by T. Jazedje et al. addresses this issue at the preclinical stage, showing in a murine breast adenocarcinoma model that mesenchymal stromal cells (MSC) may exert either pro- or antitumorigenic effects depending on the experimental condition, R. Schweizer et al. bring a more clinical perspective to this issue, presenting an interesting discussion about the possible oncogenic hazards of adipose-derived MSC in breast cancer patients subjected to breast reconstruction after mastectomy. Finally, due to their tumor homing properties, the use of stem cells as vehicles to deliver suicide genes is a strategy highly pursued in cancer gene therapy. However, low delivery efficiency and off-target effects are common limitations of current expression systems. In the original article by Y. Luo et al., the authors elegantly characterize a novel inducible transgene expression system based on the action of neural stem cells that could be further explored to treat malignant gliomas. In summary, the articles compiled in this special issue highlight how the growing investigation of tumor development through the lens of stem cell biologists is significantly impacting basic and translational cancer research. This interplay between stem cell and tumor biology offers an exceptional opportunity to improve our knowledge about cancer, one of the leading causes of death worldwide. Advances in this field are expected to bring significant impacts in cancer diagnosis and therapy. Oswaldo Keith Okamoto Ander Matheu Luca Magnani

Progress in understanding role of epithelial to mesenchymal transition in acquisition of stem cell properties by colorectal tumor cells

Progress in understanding role of epithelial to mesenchymal transition in acquisition of stem cell properties by colorectal tumor cells

Comparative proteome profiling of breast tumor cell lines by gel electrophoresis and mass spectrometry reveals an epithelial mesenchymal transition associated protein signature

The epithelial to mesenchymal transition (EMT) is a cellular program associated with the organ morphogenesis but also with the disease progression. EMT in the cancer field fuels neoplastic progression promoting the resistance to cell death, the resistance to chemotherapy and the acquisition of stem cell properties. Considering the crucial role of EMT in breast cancer metastasis, a better understanding of this process may provide new therapeutic options. Here, by using a proteomic approach we identified a set of proteins differentially expressed between an epithelial and a mesenchymal breast cancer cell line. The protein-protein network of these identified proteins was determined by an in silico analysis highlighting, in the EMT program, the role of proteins involved in cell adhesion, migration, and invasion, together with protein kinases involved in proliferation and survival, with many of these emerging as possible targets of novel biological agents. Finally, the pharmacological inhibition of some of these kinases was able to reverse the mesenchymal phenotype to an epithelial phenotype.