Initial Stages Of Tumorigenesis Research Articles

Α Humanized RANKL Transgenic Mouse Model of Progestin-Induced Mammary Carcinogenesis for Evaluation of Novel Therapeutics.

Receptor activator of nuclear factor-κB ligand (RANKL) is critically involved in mammary gland pathophysiology, while its pharmaceutical inhibition is being currently investigated in breast cancer. Herein, we investigated whether the overexpression of human RANKL in transgenic mice affects hormone-induced mammary carcinogenesis, and evaluated the efficacy of anti-RANKL treatments, such as OPG-Fc targeting both human and mouse RANKL or Denosumab against human RANKL. We established novel MPA/DMBA-driven mammary carcinogenesis models in TgRANKL mice that express both human and mouse RANKL, as well as in humanized humTgRANKL mice expressing only human RANKL, and compared them to MPA/DMBA-treated wild-type (WT) mice. Our results show that TgRANKL and WT mice have similar levels of susceptibility to mammary carcinogenesis, while OPG-Fc treatment restored mammary ductal density, and prevented ductal branching and the formation of neoplastic foci in both genotypes. humTgRANKL mice also developed MPA/DMBA-induced tumors with similar incidence and burden to those of WT and TgRANKL mice. The prophylactic treatment of humTgRANKL mice with Denosumab significantly prevented the rate of appearance of mammary tumors from 86.7% to 15.4% and the early stages of carcinogenesis, whereas therapeutic treatment did not lead to any significant attenuation of tumor incidence or tumor burden compared to control mice, suggesting the importance of RANKL primarily in the initial stages of tumorigenesis. Overall, we provide unique genetic tools for investigating the involvement of RANKL in breast carcinogenesis, and allow the preclinical evaluation of novel therapeutics that target hormone-related breast cancers.

Abstract 795: GDF15, the putative mitokine factor, promotes tumor progression in thyroid cancer via STAT3 regulation

Abstract Purpose: As mitochondrial dysfunction is an integral component during the initial stages of tumorigenesis, mechanistic insight on the interaction between these two pathways may lead to novel therapeutic approaches. The aim of this study is to investigate the role of GDF15, the putative mitokine factor in thyroid tumorigenesis. Methods: Expression levels of GDF15 was investigated using The Cancer Genome Atlas database. The role of GDF15 in tumor aggressiveness was investigated by analyzing the effects of knock-down of GDF15 on proliferation, migration, and invasion of thyroid cancer cells. Serum levels of GDF15 were measured in healthy subjects and patients with papillary thyroid cancer (PTC), and expression of GDF15 by IHC was investigated in PTC samples. To investigate the exact mechanism of GDF15 in thyroid cancer, both IPA analysis and GSEA analysis were performed using TCGA database and RNA seq data from Chungnam national university hospital. Results: TCGA data revealed the higher expression of GDF15 in tumor samples compared to in paired non-tumor samples. Thyroid cancer cells with knock-down of GDF15 revealed the decrease of proliferation, migration, and invasion compared to control cells via regulation of STAT3. Higher serum levels of GDF15 were found in patients with thyroid cancer than in control participants, and were significantly associated with tumor stage, lymphovascular invasion, and recurrence. In addition, patients with high GDF15 expression by IHC revealed the aggressive phenotype compared to patients with low GDF15 expression. Conclusions: GDF15 is a new biomarker for predicting tumor progression, and therapies targeting GDF15 may be effective for treating thyroid cancer. Citation Format: Yea Eun Kang, Jae Won Chang, Lihua Liu, Kyoungmin Lee, Mi Ae Lim, Seung-Nam Jung, Chan Oh, Ho-Ryun Won, Minho Shong, Bon Seok Koo. GDF15, the putative mitokine factor, promotes tumor progression in thyroid cancer via STAT3 regulation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 795.

Oncogenic Notch Triggers Neoplastic Tumorigenesis in a Transition-Zone-like Tissue Microenvironment

During the initial stages of tumorigenesis, the tissue microenvironment where the pro-tumor cells reside plays a crucial role in determining the fate of these cells. Transition zones, where two types of epithelial cells meet, are high-risk sites for carcinogenesis, but the underlying mechanism remains largely unclear. Here, we show that persistent upregulation of Notch signaling induces neoplastic tumorigenesis in a transition zone between the salivary gland imaginal ring cells and the giant cells in Drosophila larvae. In this region, local endogenous JAK-STAT and JNK signaling creates a tissue microenvironment that is susceptible to oncogenic-Notch-induced tumorigenesis, whereas the rest of the salivary gland imaginal ring is refractory to Notch-induced tumor transformation. JNK signaling activates a matrix metalloprotease (MMP1) to promote Notch-induced tumorigenesis at the transition zone. These findings illustrate the significance of local endogenous inflammatory signaling in primary tumor formation.

Hijacking the Hexosamine Biosynthetic Pathway to Promote EMT-Mediated Neoplastic Phenotypes.

The epithelial–mesenchymal transition (EMT) is a highly conserved program necessary for orchestrating distant cell migration during embryonic development. Multiple studies in cancer have demonstrated a critical role for EMT during the initial stages of tumorigenesis and later during tumor invasion. Transcription factors (TFs) such as SNAIL, TWIST, and ZEB are master EMT regulators that are aberrantly overexpressed in many malignancies. Recent evidence correlates EMT-related transcriptomic alterations with metabolic reprograming in cancer. Metabolic alterations may allow cancer to adapt to environmental stressors, supporting the irregular macromolecular demand of rapid proliferation. One potential metabolic pathway of increasing importance is the hexosamine biosynthesis pathway (HBP). The HBP utilizes glycolytic intermediates to generate the metabolite UDP–GlcNAc. This and other charged nucleotide sugars serve as the basis for biosynthesis of glycoproteins and other glycoconjugates. Recent reports in the field of glycobiology have cultivated great curiosity within the cancer research community. However, specific mechanistic relationships between the HBP and fundamental pathways of cancer, such as EMT, have yet to be elucidated. Altered protein glycosylation downstream of the HBP is well positioned to mediate many cellular changes associated with EMT including cell–cell adhesion, responsiveness to growth factors, immune system evasion, and signal transduction programs. Here, we outline some of the basics of the HBP and putative roles the HBP may have in driving EMT-related cancer processes. With novel appreciation of the HBP’s connection to EMT, we hope to illuminate the potential for new therapeutic targets of cancer.

Tumor Microenvironment Versus Cancer Stem Cells in Cholangiocarcinoma: Synergistic Effects?

Cholangiocarcinoma (CCAs) may be defined as tumors that derived from the biliary tree with the differentiation in the biliary epithelial cells. This tumor is malignant, extremely aggressive with a poor prognosis. It can be treated surgically and its pathogenesis is poorly understood. The tumor microenvironment (TME) is a very important factor in the regulation of tumor angiogenesis, invasion, and metastasis. Besides cancer stem cells (CSCs) can modulate tumor growth, stroma formation, and migratory capability. The initial stage of tumorigenesis is characterized by genetic mutations and epigenetic alterations due to intrinsic factors which lead to the generation of oncogenes thus inducing tumorigenesis. CSCs may result from precancerous stem cells, cell de-differentiation, normal stem cells, or an epithelial-mesenchymal transition (EMT). CSCs have been found in the cancer niche, and EMT may occur early within the tumor microenvironment. Previous studies have demonstrated evidence of cholangiocarcinoma stem cells (CD133, CD24, EpCAM, CD44, and others) and the presence of these markers has been associated with malignant potential. The interaction between TME and cholangiocarcinoma stem cells via signaling mediators may create an environment that accommodates tumor growth, yielding resistance to cytotoxic insults (chemotherarapeutic). While progress has been made in the understanding of the mechanisms, the interactions in the tumorigenic process still remain a major challenge. Our review, addresses recent concepts of TME-CSCs interaction and will emphasize the importance of early detection with the use of novel diagnostic mechanisms such as CCA-CSC biomarkers and the importance of tumor stroma to define new treatments. J. Cell. Physiol. 231: 768-776, 2016. © 2015 Wiley Periodicals, Inc.

Abstract 1323: Lack of cyclin D3 enhances the CDK6-dependent skin tumor susceptibility to malignant progression

Abstract The Cyclin-Dependent Kinases (CDKs) are a family of essential serine-threonine kinases that are cell cycle regulated by association with cyclins at specific time-points of the cell cycle. Specifically, D-type cyclins form complexes and activate CDK6 and CDK4. We have previously established that transgenic expression of CDK6 in mouse epidermis (K5CDK6 mice) alter an early stage of tumorigenesis leading to strong reduction of the multiplicity and incidence of skin tumors. Our previous results have shown that CDK6 preferentially binds to cyclin D3, which overexpression also decreases tumor development in mouse epidermis. Thus, we hypothesized that the association of CDK6/cyclin D3 and its enzymatic activity negatively impacts tumor development. To test this idea, we developed K5CDK6/Cyclin D3-/- mutant mice and subjected them to the two-stage carcinogenesis protocol. Contrary to our hypothesis, lack of cyclin D3 expression does not affect the tumor repression activity of CDK6 since both, K5CDK6 and K5CDK6/cyclin D3-/- mice, exhibit a severe reduction in the multiplicity and incidence of skin tumors compared to wild-type siblings. However, histopathological analysis of skin tumors revealed that 60% of the skin tumors in K5CDK6/cyclin D3-/- mice were classified as squamous-cell carcinomas I (SCC) with a variable degree of squamous differentiation; whereas wild type, K5CDK6 and cyclin D3-/- siblings exhibit benign tumors with no significant changes in the rate of malignant progression. Consistent with the malignant nature of the K5CDK6/cyclin D3-/- tumors, lack or reduction of cyclin D3 levels within the context of CDK6 overexpression (K5CDK6/D3-/- and K5CDK6/cyclin D3+/-) lead to 7-fold increase in tumor size compared to K5CDK6, cyclin D3-/- and wild-type tumors. Biochemical analysis of epidermal tumors shows that cyclin D3 ablation results in a rise of cyclin D1 protein levels. These data suggest that though cyclin D3 expression does not alter the tumor suppressive role of CDK6 at early stages of tumorigenesis, it does affect the expression of cyclin D1 resulting in the increased rate of malignant progression. Consistent with these studies, we have shown that elevated activity of CDK4/cyclin D1 complex results in an elevated number of SCC. We conclude that CDK6 activity can prevent tumor formation during the initial stages of tumorigenesis in a cyclin D3-independent manner and likely Rb-independent as well (Kollman et al, 2013). However, variations in the expression of cyclin D3 affects cyclin D2 (Rojas et al, 2007) and/or cyclin D1 levels, directly altering the susceptibility of CDK6 expressing tumors to malignant conversion. Therefore, therapies directed to reduce D-type cyclins expression should be considered within the context of the expression level of CDK6 and CDK4 to define the potential effect in the malignant progression of skin tumors. Supported by NIH grant CA116328. Note: This abstract was not presented at the meeting. Citation Format: Sung Hyun Lee, Xian Wang, Marcelo L. Rodriguez-Puebla. Lack of cyclin D3 enhances the CDK6-dependent skin tumor susceptibility to malignant progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1323. doi:10.1158/1538-7445.AM2014-1323

Adoptive transfer of regulatory T cells promotes intestinal tumorigenesis and is associated with decreased NK cells and IL‐22 binding protein

High number of regulatory T cells (Tregs), both circulating and at the tumor site, often indicates a poor prognosis in CRC patient's possibly impairing natural killer (NK) cell function. To determine the role of Tregs in CRC development and their effects on NK cells, we created novel transgenic Rag-Apc mice that lack T cells and develop spontaneous intestinal tumors, and we adoptively transferred Tregs or transiently depleted NK cells during initial stages of tumorigenesis. In 6-weeks old Rag-Apc mice containing microscopic intestinal tumors adoptive transfer of Tregs or transient NK cell depletion dramatically associated with an increase in intestinal tumor multiplicity and tumor size, with significantly decreased survival rates. Importantly, Treg transfer increased small intestinal polyp formation up to 65% (P < 0.0005) and increased colon tumors multiplicities by 84% (P < 0.0001) with a significant decrease in NK cells as compared to control mice. Similarly, in NK depleted mice, colon tumor multiplicities increased up to 40% and small intestinal polyp formation up to 60% (P < 0.0001). Treg transfer or NK cell transient depletion markedly increased interleukin (IL)-22 systemically and the inflammatory signaling molecules P2X7R, and STAT3 in the tumors; and impaired production of the tumor suppressor interferon (IFN)-γ systemically. Notably, IL-22 binding protein (IL-22 BP) was associated with NKs and a significant decrease was seen at the tumor site in mice adoptively transferred with Tregs or depleted of NK cells. Our results suggest that adoptive transfer of Tregs aggressively promote intestinal tumorigenesis by decreasing NK cell number and activity by modulating IL-22 BP.

Identification of new pro-apoptotic inhibitors of HSP70

Oridonin is a very promising anti-cancer ent-kaurane diterpene.1 – 4 Recently we have demonstrated that the pro-apoptotic activity of oridonin depends on its ability inhibit the activity of HSP70.5 It is a member of a ubiquitously expressed family of molecular chaperones involved in de novo protein folding, subcellular trafficking, proteasome-mediated degradation, and autophagy. At the initial stages of tumorigenesis, HSP70 has been shown to protect the cells undergoing transformation from oncogenic stress induced by overexpression of oncogenes. HSP70 over-expression has been routinely associated with poor prognosis in multiple forms of cancer and is thought to provide a survival advantage to cancer cells interacting with multiple cellular pathways.6

Serum protein signature may improve detection of ductal carcinoma in situ of the breast

Ductal carcinoma in situ (DCIS) of the breast is part of a spectrum of preinvasive lesions that originate within normal breast tissue and progress to invasive breast cancer. The detection of DCIS is important for the reduction of mortality from breast cancer, but the diagnosis of preinvasive breast tumors is hampered by the lack of an adequate detection method. To identify the changes in protein expression during the initial stage of tumorigenesis and to identify the presence of new DCIS markers, we analysed serum from 60 patients with breast cancer and 60 normal controls using mass spectrometry. A 23-protein index was generated that correctly distinguishes the DCIS and control groups with sensitivities and specificities in excess of 80% in two independent cohorts. Two candidate peptides were purified and identified as platelet factor 4 (PF-4) and complement C3a(desArg) anaphylatoxin (C3a(desArg)) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In an independent serum set of 165 patients, PF-4 and C3a(desArg) were significantly upregulated in DCIS compared with non-cancerous controls, as validated using western blot and enzyme-linked immunosorbent assay. We conclude that our serum protein-based test, used in conjunction with image-based screening practices, could improve the sensitivity and specificity of breast cancer detection.

In Pursuit of Leucine-Rich Repeat-Containing G Protein-Coupled Receptor-5 Regulation and Function in the Uterus

Leucine-rich repeat-containing G protein-coupled receptor (LGR)-5 is a recently identified marker of stem cells in adult intestinal epithelium and hair follicles. Because of this characteristic, we studied the status of Lgr5 expression in the mouse uterus under various conditions. Lgr5 is highly expressed in the uterine epithelium of immature mice and is dramatically down-regulated after the mice resume estrous cycles. Surprisingly, whereas its expression is up-regulated in uteri of ovariectomized mice, the expression is down-regulated by estrogen and progesterone via their cognate nuclear receptors, estrogen receptor-alpha and progesterone receptor, respectively. Using a mouse endometrial cancer model, we also found that Lgr5 is highly expressed in the epithelium during the initial stages of tumorigenesis but is remarkably down-regulated in fully developed tumors. Lgr5 is a downstream target of Wnt signaling in the intestine. Genetic evidence shows that either excessive or absence of Wnt signaling dampens Lgr5 expression in the uterus. Collectively, our results show that Lgr5 expression in the mouse uterine epithelium is unique and dynamically regulated under various physiological and pathological states of the uterus, suggesting that this orphan receptor has important functions in uterine biology. However, identifying definitive uterine function of LGR5 will require further investigation using conditional deletion of uterine Lgr5 because systemic deletion of this gene is neonatally lethal.

Ionizing Radiation Predisposes Nonmalignant Human Mammary Epithelial Cells to Undergo Transforming Growth Factor β–Induced Epithelial to Mesenchymal Transition

Abstract Transforming growth factor β1 (TGFβ) is a tumor suppressor during the initial stage of tumorigenesis, but it can switch to a tumor promoter during neoplastic progression. Ionizing radiation (IR), both a carcinogen and a therapeutic agent, induces TGFβ activation in vivo. We now show that IR sensitizes human mammary epithelial cells (HMEC) to undergo TGFβ-mediated epithelial to mesenchymal transition (EMT). Nonmalignant HMEC (MCF10A, HMT3522 S1, and 184v) were irradiated with 2 Gy shortly after attachment in monolayer culture or treated with a low concentration of TGFβ (0.4 ng/mL) or double treated. All double-treated (IR + TGFβ) HMEC underwent a morphologic shift from cuboidal to spindle shaped. This phenotype was accompanied by a decreased expression of epithelial markers E-cadherin, β-catenin, and ZO-1, remodeling of the actin cytoskeleton, and increased expression of mesenchymal markers N-cadherin, fibronectin, and vimentin. Furthermore, double treatment increased cell motility, promoted invasion, and disrupted acinar morphogenesis of cells subsequently plated in Matrigel. Neither radiation nor TGFβ alone elicited EMT, although IR increased chronic TGFβ signaling and activity. Gene expression profiling revealed that double-treated cells exhibit a specific 10-gene signature associated with Erk/mitogen-activated protein kinase (MAPK) signaling. We hypothesized that IR-induced MAPK activation primes nonmalignant HMEC to undergo TGFβ-mediated EMT. Consistent with this, Erk phosphorylation was transiently induced by irradiation and persisted in irradiated cells treated with TGFβ, and treatment with U0126, a MAP/Erk kinase (MEK) inhibitor, blocked the EMT phenotype. Together, these data show that the interactions between radiation-induced signaling pathways elicit heritable phenotypes that could contribute to neoplastic progression. [Cancer Res 2007;67(18):8662–70]

Erythropoietin Blockade Inhibits the Induction of Tumor Angiogenesis and Progression

BackgroundThe induction of tumor angiogenesis, a pathologic process critical for tumor progression, is mediated by multiple regulatory factors released by tumor and host cells. We investigated the role of the hematopoietic cytokine erythropoietin as an angiogenic factor that modulates tumor progression.Methodology/Principal FindingsFluorescently-labeled rodent mammary carcinoma cells were injected into dorsal skin-fold window chambers in mice, an angiogenesis model that allows direct, non-invasive, serial visualization and real-time assessment of tumor cells and neovascularization simultaneously using intravital microscopy and computerized image analysis during the initial stages of tumorigenesis. Erythropoietin or its antagonist proteins were co-injected with tumor cells into window chambers. In vivo growth of cells engineered to stably express a constitutively active erythropoietin receptor EPOR-R129C or the erythropoietin antagonist R103A-EPO were analyzed in window chambers and in the mammary fat pads of athymic nude mice. Co-injection of erythropoietin with tumor cells or expression of EPOR-R129C in tumor cells significantly stimulated tumor neovascularization and growth in window chambers. Co-injection of erythropoietin antagonist proteins (soluble EPOR or anti-EPO antibody) with tumor cells or stable expression of antagonist R103A-EPO protein secreted from tumor cells inhibited angiogenesis and impaired tumor growth. In orthotopic tumor xenograft studies, EPOR-R129C expression significantly promoted tumor growth associated with increased expression of Ki67 proliferation antigen, enhanced microvessel density, decreased tumor hypoxia, and increased phosphorylation of extracellular-regulated kinases ERK1/2. R103A-EPO antagonist expression in mammary carcinoma cells was associated with near-complete disruption of primary tumor formation in the mammary fat pad.Conclusions/SignificanceThese data indicate that erythropoietin is an important angiogenic factor that regulates the induction of tumor cell-induced neovascularization and growth during the initial stages of tumorigenesis. The suppression of tumor angiogenesis and progression by erythropoietin blockade suggests that erythropoietin may constitute a potential target for the therapeutic modulation of angiogenesis in cancer.

Dietary fat and fiber differentially alter intracellular second messengers during tumor development in rat colon.

The effect of fat, fiber and carcinogen on colonic epithelial intracellular second messengers 1,2-diacyl-sn-glycerol (DAG), ceramide, and the steady-state level of phospholipase C (PLC-gamma1) was determined in 160 male Sprague-Dawley rats (10 rats per group). The study was a 2 x 2 x 2 x 2 factorial design with two types of fat (corn oil or fish oil), two types of fiber (cellulose or pectin), two injected subgroups (with or without azoxymethane (AOM), and two time points (15 and 37 weeks). At the final time point (37 weeks) there were an additional 20 rats per diet in each of the carcinogen-treated groups for tumor analyses only (n = 80), for a total of 240 animals in the entire study. At each time point (15 and 37 weeks), 80 rats were killed and colonic mucosa obtained for DAG, ceramide and PLC-gamma1 assays. At the first time point (15 weeks), there was no microscopic evidence of tumors. At the final time point (37 weeks), fish oil resulted in a lower proportion of animals with adenocarcinomas relative to corn oil feeding (56.1 % versus 69.6 %, P < 0.05). There was no significant main effect of fiber on the percentage of animals with tumors. At 15 weeks post-injection, AOM injected animals fed corn oil-containing diets had a significantly (P < 0.001) higher DAG mass and steady-state levels of PLC-gamma1 compared with AOM-injected animals fed fish oil and saline injected rats on all diets. Animals fed corn oil diets also had a significantly (P < 0.01) elevated mucosal ceramide mass compared with fish oil fed animals. Moreover, rats injected with AOM had a significantly (P < 0.02) elevated colonic mucosal DAG/ceramide ratio versus saline injected animals. In contrast, dietary fiber had no effect on any of the parameters measured at 15 weeks. However, at 37 weeks post-injection, dietary fiber significantly altered DAG (P < 0.02), and PLC-gamma1 expression (P < 0.05) in the absence of an effect on tumor incidence. These data demonstrate that the ability of dietary fish oil to reduce experimental colon carcinogenesis may be mediated by changes in colonic intracellular mediators during the initial stages of tumorigenesis.

Thymoma: tumour type related to expression of epidermal growth factor (EGF), EGF-receptor, p53, v-erb B and ras p21.

As clinicopathological features may not be sufficient to predict the progression of thymoma, we have carried out what we believe to be the first immunohistochemical study describing the relationship between the different types of thymoma and the tumour stage, on the one hand, and the expression of epidermal growth factor (EGF), EGF-receptor (EGFR), p53, v-erb B and ras p21, on the other. The positive rates versus histological types and Masaoka's clinical stages in the 47 cases were as follows: p53 (non-invasive thymoma: 41.7%; malignant thymoma category I: 82.4%; malignant thymoma category II: 83.3%), EGF (non-invasive thymoma: 4.2%; malignant thymoma category I: 11.8%; malignant thymoma category II: 33.3%) and EGFR (non-invasive thymoma: 8.3%; malignant thymoma category I: 35.3%; malignant thymoma category II: 66.7%); p53 (stages I and II: 51.7%; stages III and IV: 77.8%), EGF (stages I and II: 3.4%; stages III and IV: 22.2%) and EGFR (stages I and II: 13.8%; stages III and IV: 44.4%). These data suggest that p53 may be implicated in the initial stages of tumorigenesis and that increased expression of EGF and EGFR may play a role in thymoma progression.