Promote Cancer Initiation Research Articles

Tumor-associated macrophages promote the metastatic potential of thyroid papillary cancer by releasing CXCL8

Tumor-associated macrophages (TAMs) can promote cancer initiation and progression by releasing cytokines. Previously, we have found the density of TAMs correlated with lymph node metastasis in papillary thyroid carcinoma (PTC). However, the mechanisms of how TAMs promote PTC progression remain unclear. In this study, we first showed that the TAMs density in the tumor core was associated with progressive PTC features and TAMs conditioned medium enhanced PTC cells invasion. Cytokine profiling identified a mixed M1/M2 phenotype and CXCL8 was the most consistently abundant cytokine in PTC-derived TAMs. CXCL8 receptors, CXCR1 and CXCR2, were positively stained in PTC cell lines and tissues, though no association with lymph node metastasis or extrathyroid extension. PTC cell invasion was abrogated by anti-CXCL8-neutralizing antibody, whereas addition of exogenous recombinant human CXCL8 enhanced the invasiveness. More importantly, CXCL8 promoted PTC metastasis in vivo. No difference was found for TAMs-derived CXCL8 expression in patients with and without lymph node metastasis or extrathyroid extension. These findings indicated that TAMs may facilitate PTC cell metastasis through CXCL8 and its paracrine interaction with CXCR1/2.

Chapter Two - Autophagy and Cancer Metabolism

The metabolism of malignant cells is profoundly altered in order to maintain their survival and proliferation in adverse microenvironmental conditions. Autophagy is an intracellular recycling process that maintains basal levels of metabolites and biosynthetic intermediates under starvation or other forms of stress, hence serving as an important mechanism for metabolic adaptation in cancer cells. Although it is widely acknowledged that autophagy sustains metabolism in neoplastic cells under duress, many questions remain with regard to the mutual relationship between autophagy and metabolism in cancer. Importantly, autophagy has often been described as a "double-edged sword" that can either impede or promote cancer initiation and progression. Here, we overview such a dual function of autophagy in tumorigenesis and our current understanding of the coordinated regulation of autophagy and cancer cell metabolism in the control of tumor growth, progression, and resistance to therapy.

RhoB Promotes Cancer Initiation by Protecting Keratinocytes from UVB-Induced Apoptosis but Limits Tumor Aggressiveness

The role of UVB-induced apoptosis in the formation of squamous cell carcinoma (SCC) is recognized. We previously identified the small RhoB (Ras homolog gene family, member B) GTPase, an early response gene to cellular stress, as a critical protein controlling apoptosis of human keratinocytes after UVB exposure. Here we generated SKH1 (hairless immunocompetent mouse) mice invalidated for RhoB to evaluate its role in UVB-induced skin carcinogenesis in vivo. We show that rhob-/- mice have a lower risk of developing UVB-induced keratotic tumors and actinic keratosis that is associated with a higher sensitivity of UVB-exposed keratinocytes to apoptosis. We extend this observation to primary cultures of normal human keratinocytes in which RhoB was downregulated with small interfering RNA (siRNA) and further show that the hypersensitivity to apoptosis depends on B-cell lymphoma 2 (Bcl-2) downregulation. In rhob-/- mice, the UVB-induced tumors were preferentially undifferentiated and highly proliferative. Finally, we show in humans an almost constant loss of RhoB expression in undifferentiated SCCs. These undifferentiated and RhoB-deficient tumors have elevated phosphorylated histone H2AX (γH2AX) and 53BP1, two markers of DNA double-strand breaks. Together, our results indicate that UVB-induced RhoB expression participates in in vivo SCC initiation by increasing keratinocyte survival. Conversely, RhoB may limit tumor aggressiveness as loss of RhoB expression in tumor cells is associated with tumor progression.

Targeting Tumor-Infiltrating Macrophages to Combat Cancer

Tumor-associated macrophages are one of the major constituents of tumor stroma in many solid tumors and there is compelling preclinical and clinical evidence that macrophages promote cancer initiation and malignant progression. Therefore, these cells represent potential targets for therapeutic benefit. In this review, we will summarize macrophage phenotypic heterogeneity, the current understanding of how tumors take advantage of macrophage plasticity to generate immunosuppression, and how manipulation of specific macrophage populations can be used for therapeutic purposes through translational approaches.

Identification of FOXM1‐induced epigenetic markers for head and neck squamous cell carcinomas

Epigenetic reprogramming of the methylome has been implicated in all stages of cancer evolution. It is now well accepted that cancer cells exploit epigenetic reprogramming, a mechanism that regulates stem/progenitor cell renewal and differentiation, to promote cancer initiation and progression. The oncogene FOXM1 has been implicated in all major forms of human cancer. We have recently shown that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a number of FOXM1-induced epigenetic markers. Differential promoter methylation and gene expression in clinical specimens were measured using commercially available bisulfite conversion kits and absolute quantitative PCR, respectively. Here, we investigated 8 FOXM1-induced differentially methylated genes, SPCS1, FLNA, CHPF, GLT8D1, C6orf136, MGAT1, NDUFA10, and PAFAH1B3, using human head and neck tissue specimens donated by 2 geographically independent patient cohorts from Norway and the United Kingdom. Two genes (GLT8D1 and C6orf136) were found to be differentially expressed in head and neck squamous cell carcinomas (HNSCCs). Using methylation-specific quantitative PCR, we confirmed that the promoters of GLT8D1 and C6orf136 were hypo- and hypermethylated, respectively, in HNSCC tissues. Given that epigenetic change precedes gene expression, methylation status of candidate genes identified from this study may represent a signature of premalignancy, rendering them potentially useful predictive biomarkers for precancer screening and/or therapeutic targets for cancer prevention.

Is Upregulation of BCL2 a Determinant of Tumor Development Driven by Inactivation of CDH1/E-Cadherin?

Inactivation of CDH1, encoding E-cadherin, promotes cancer initiation and progression. According to a newly proposed molecular mechanism, loss of E-cadherin triggers an upregulation of the anti-apoptotic oncoprotein BCL2. Conversely, reconstitution of E-cadherin counteracts overexpression of BCL2. This reciprocal regulation is thought to be critical for early tumor development. We determined the relevance of this new concept in human infiltrating lobular breast cancer (ILBC), the prime tumor entity associated with CDH1 inactivation. BCL2 expression was examined in human ILBC cell lines (IPH-926, MDA-MB-134, SUM-44) harboring deleterious CDH1 mutations. To test for an intact regulatory axis between E-cadherin and BCL2, wild-type E-cadherin was reconstituted in ILBC cells by ectopic expression. Moreover, BCL2 and E-cadherin were evaluated in primary invasive breast cancers and in synchronous lobular carcinomas in situ (LCIS). MDA-MB-134 and IPH-926 showed little or no BCL2 expression, while SUM-44 ILBC cells were BCL2-positive. Reconstitution of E-cadherin failed to impact on BCL2 expression in all cell lines tested. Primary ILBCs were almost uniformly E-cadherin-negative (97%) and were frequently BCL2-negative (46%). When compared with an appropriate control group, ILBCs showed a trend towards an increased frequency of BCL2-negative cases (P = 0.064). In terminal duct-lobular units affected by LCIS, the E-cadherin-negative neoplastic component showed a similar or a reduced BCL2-immunoreactivity, when compared with the adjacent epithelium. In conclusion, upregulation of BCL2 is not involved in lobular breast carcinogenesis and is unlikely to represent an important determinant of tumor development driven by CDH1 inactivation.

Virus induced inflammation and cancer development

Chronic inflammation as a result of viral infection significantly increases the likelihood of cancer development. A handful of diverse viruses have confirmed roles in cancer development and progression, but the list of suspected oncogenic viruses is continually growing. Viruses induce cancer directly and indirectly, by activating inflammatory signalling pathways and cytokines, stimulating growth of infected cells and inhibiting apoptosis. Although oncogenic viruses induce inflammation by various mechanisms, it is generally mediated by the MAPK, NFκB and STAT3 signalling pathways. This review will explore the unique mechanisms by which different oncogenic viruses induce inflammation to promote cancer initiation and progression.

Abstract B28: Signaling by PDGF-CC in the maintenance of a protumorigenic breast cancer microenvironment

Abstract It is increasingly appreciated that cancer results from the concerted performance of genetically altered tumor cells interacting with ostensibly normal cells in the microenvironment. As the cancer progresses, the surrounding stroma co-evolves into an activated state through continuous paracrine communication, thus creating a dynamic signaling circuitry that promotes cancer initiation and growth, and ultimately leads to a fatal disease. We aim to molecularly map the support functions performed by the various cell types comprising the tumor stroma. Specifically we will focus on, cancer-associated fibroblasts (CAFs), which are known to support many aspects of tumor progression. Based on our previous finding that paracrine signaling by Platelet-derived growth factor (PDGF)-CC serves to recruit CAFs into tumors we have generated Pdgfc-deficient genetically engineered mouse models of breast cancer. Our results show a significant reduction in the tumor burden followed by prolonged survival in the absence of PDGF-CC, in agreement with the recent finding that PDGF-CC expression is an indicator of poor prognosis for breast cancer patients. Upon transplantation of small tumor pieces from wildtype and Pdgfc-deficient tumors respectively into the mammary fatpad of wildtype mice, Pdgfc-deficient tumors show substantial growth retardation with massive hemorrhage and necrosis, indicative of malfunctional vessels and insufficient blood supply. qPCR arrays on total RNA from wildtype and Pdgfc-deficient tumors respectively, suggest that lack of PDGF-CC causes perturbed differentiation yielding a less advanced tumor. Furthermore, we are currently investigating the differences observed in the recruited fibroblasts and/or MSCs in wildtype and Pdgfc-deficient tumors, by performing proteomics on isolated PDGFR-α positive cells. Tumor morphology in combination with proteomics suggests that the lack of PDGF-CC causes an impaired migration and/or guidance of the recruited fibroblasts from the periphery of the tumor. Thus, our studies suggest a novel role for PDGF-CC in creating a pro-tumorigenic microenvironment and identify PDGF-CC as a valid and promising therapeutic target in breast cancer. Citation Format: Pernilla Roswall, Kristian Haller, Annika Petersson, Charlotte Anderberg, Hong Li, Jens-Henrik Norum, Rune Toftgård, Ulf Eriksson, Kristian Pietras. Signaling by PDGF-CC in the maintenance of a protumorigenic breast cancer microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B28.

MiRNAs in cancer: Non-coding RNAs as appealing biomarkers for malignancy

It is estimated that up to 50% of all human proteincoding genes are regulated by miRNAs. This family of non-coding RNAs, with ∼1500 members reported up-to-date, can impact a variety of cellular processes including cellular proliferation, differentiation and apoptosis. It is not surprising that deregulated expression of selected miRNAs can thus promote cancer initiation and progression. Several miRNAs with oncogenic properties, termed oncomirs, as well as miRNAs with tumor suppressive capabilities have been identified in different malignancies and this list is growing at a rapid pace. MiRNA-based therapies are attractive partly due to the fact that these molecules can target multiple genes in different signaling pathways simultaneously. In addition to their therapeutic potential, miRNAs are released into the circulation and measurement of such species in plasma and serum samples highlights the possibility of leveraging these molecules as potential biomarkers for early cancer detection, prognostic assessment and evaluation of therapeutic response in cancer patients. In this issue of Cancer Biomarkers, Lodewijk et al. discuss differentially expressed miRNAs in subtypes of thyroid cancer: papillary, follicular, anaplastic and medullary thyroid carcinomas. A special interest is placed on the diagnostic potential of miRNAs in this malignancy. The current method utilized to characterize thyroid nodules consisting of fine needle aspiration biopsies is described. Unfortunately, 30% of such biopsies are inconclusive and a hemithyroidectomy is required. Their article thus delves into the potential usefulness of identifying amiRNA signature that would help avoid such a procedure and assist clinicians in discriminating between tumor and benign tissues as well as between tumor types. Similarly, Hogan et al. describe in this issue a need for the identification of novel biomarkers in the early detection of colorectal cancer (CRC). 5-year patient survival is over 90% when detected at an early stage compared with less than 10% survival for stage IV patients. While advantages exist with the currentmethods employed for CRC detection, sensitivity and specificity challenges are discussed and the diagnostic potential of miRNAs is assessed. MiRNA stability and their presence in body fluids including feces make them attractive molecules for the development of noninvasive detection methods for this malignancy. Much effort is also directed towards the identification of a circulating miRNA signature in glioblastoma multiforme (GBM), an aggressive type of brain tumor with dismal prognosis. In this issue, Odjele et al. review the progress made in recent years on identifying miRNAs that would assist clinicians in diagnosing this malignancy. MiRNA levels of miR-128 and miR342-3p vary significantly in blood samples of GBM patients when compared with healthy subjects and are thus emerging as early favorites in the search for a GBM-associated miRNA signature. The article also looks at studies recently undertaken in GBM cell and rodent models that showed promising results on the use

Stromal cells in tumor microenvironment and breast cancer

Cancer is a systemic disease encompassing multiple components of both tumor cells themselves and host stromal cells. It is now clear that stromal cells in the tumor microenvironment play an important role in cancer development. Molecular events through which reactive stromal cells affect cancer cells can be defined so that biomarkers and therapeutic targets can be identified. Cancer-associated fibroblasts (CAFs) make up the bulk of cancer stroma and affect the tumor microenvironment such that they promote cancer initiation, angiogenesis, invasion, and metastasis. In breast cancer, CAFs not only promote tumor progression but also induce therapeutic resistance. Accordingly, targeting CAFs provides a novel way to control tumors with therapeutic resistance. This review summarizes the current understandings of tumor stroma in breast cancer with a particular emphasis on the role of CAFs and the therapeutic implications of CAFs. In addition, the effects of other stromal components such as endothelial cells, macrophages, and adipocytes in breast cancer are also discussed. Finally, we describe the biologic markers to categorize patients into a specific and confirmed subtype for personalized treatment.

Abstract B34: Receptor tyrosine kinases (RTKs) profiling and its role in advanced gastric cancer.

Abstract Background: Receptor tyrosine kinase (RTK) activation is the starting point of intracellular signaling cascades that control cellular process such as proliferation, differentiation, migration and survival and aberrant RTK activation may promote cancer initiation and progression. The purpose of this study was to determine the frequency and the influence of major RTKs including EGFR, HER-2, c-MET, and FGFR-2 expression on the outcomes of gastric cancer patients. Methods: Between January 2000 and December 2004, 122 patients with advanced gastric cancer in our institute were selected. All patients underwent gastrectomy and D2 lymph node dissection with a curative aim and pathologically confirmed N3 stage without distant metastasis. Formalin-fixed paraffin-embedded (FFPE) tissue specimens were examined for the expression of EGFR, HER-2, c-MET, FGFR-2 by immunohistochemistry (IHC). The score for expression was assessed by the independent pathologist with weighted histoscore method. We defined positive result as the expression of 3+ in IHC for each RTK. Results: Among 4 RTKs, c-MET expression showed highest frequency as 31 patients (25.4%) followed by FGFR-2 (13; 11.2%), HER-2 (10; 8.5%) and EGFR (6; 5.0%). The majority of samples (58.2%) were negative (wild type) for all four markers. Fourteen patients (11.5%) showed co-expression for these RTKs; one patient was quadruple positive, and the others showed double positivity for c-MET and one of 3 other markers. There were no significant correlations between each biomarker and clinic-pathologic parameters such as patient's gender, differentiation, T stage, lymphovascular invasion, Lauren classification or the number of LN metastasis. With the follow-up period of 21.5 months (0.5–115.7), median overall survival of all patients was 20.9 months (95% CI, 15.2–26.6) and median disease-free survival (DFS) was 17.1 months (95% CI, 9.4–24.8). Based on the status of each receptor, survival outcome was only associated with c-MET over-expression; median DFS of 10.8 months in c-MET positive patients and 22.0 months in c-MET negative patients (p=0.04). Median DFS in patients with co-expression of RTKs showed poorer trend than single marker positive group and wild type group (10.8m, 15.7m, and 19.4m, respectively; p=0.27). Conclusion: We evaluated the expression pattern with four major RTKs in relatively homogenous subgroup of gastric cancer patients with pathologic N3. c-MET over-expression could be a prognostic biomarker for DFS in these patients, suggesting the significant role of c-met in gastric cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B34.

Cancer. Aneuploidy drives a mutator phenotype in cancer.

An abnormal chromosome number alone can induce mutations that may promote cancer initiation or progression.

The Role of Pea3 Group Transcription Factors in Esophageal Squamous Cell Carcinoma

The transcription factors Pea3, Erm, and Er81 can promote cancer initiation and progression in various types of solid tumors. However, their role in esophageal squamous cell carcinoma (ESCC) has not been elucidated. In this study, we found that the expression levels of Pea3 and Erm, but not that of Er81, were significantly higher in ESCC compared with nontumor esophageal epithelium. A high level of Pea3 expression was significantly correlated with a shorter overall survival in a cohort of 81 patients with ESCC and the subgroup with N1 stage tumor (Wilcoxon-Gehan test, P = 0.016 and P = 0.001, respectively). Pea3 was overexpressed in seven ESCC cell lines compared with two immortalized esophageal cell lines. Pea3 knockdown reduced cell proliferation and suppressed nonadherent growth, migration, and invasion in ESCC cells in vitro. In addition, Pea3 knockdown in ESCC cells resulted in a down-regulation of phospho-Akt and matrix metalloproteinase 13, whereas a significant positive correlation in the expression levels was observed between Pea3 and phospho-Akt (r = 0.281, P < 0.013) and between Pea3 and matrix metalloproteinase 13 in the human specimens (r = 0.462, P < 0.001). Moreover, Pea3 modulated the sensitivity of EC109 cells to doxorubicin, probably via reduced activity of the phosphatidylinositol 3-kinase-Akt-mammalian target of Rapamycin complex 1 pathway on Pea3 knockdown. In conclusion, our results suggest that Pea3 plays an important role in the progression of ESCC.

Amplification of the 20q Chromosomal Arm Occurs Early in Tumorigenic Transformation and May Initiate Cancer

Duplication of chromosomal arm 20q occurs in prostate, cervical, colon, gastric, bladder, melanoma, pancreas and breast cancer, suggesting that 20q amplification may play a causal role in tumorigenesis. According to an alternative view, chromosomal imbalance is mainly a common side effect of cancer progression. To test whether a specific genomic aberration might serve as a cancer initiating event, we established an in vitro system that models the evolutionary process of early stages of prostate tumor formation; normal prostate cells were immortalized by the over-expression of human telomerase catalytic subunit hTERT, and cultured for 650 days till several transformation hallmarks were observed. Gene expression patterns were measured and chromosomal aberrations were monitored by spectral karyotype analysis at different times. Several chromosomal aberrations, in particular duplication of chromosomal arm 20q, occurred early in the process and were fixed in the cell populations, while other aberrations became extinct shortly after their appearance. A wide range of bioinformatic tools, applied to our data and to data from several cancer databases, revealed that spontaneous 20q amplification can promote cancer initiation. Our computational model suggests that 20q amplification induced deregulation of several specific cancer-related pathways including the MAPK pathway, the p53 pathway and Polycomb group factors. In addition, activation of Myc, AML, B-Catenin and the ETS family transcription factors was identified as an important step in cancer development driven by 20q amplification. Finally we identified 13 "cancer initiating genes", located on 20q13, which were significantly over-expressed in many tumors, with expression levels correlated with tumor grade and outcome suggesting that these genes induce the malignant process upon 20q amplification.

Research Highlights

Research Highlights

TCDD mediates inhibition of p53 and activation of ERα signaling in MCF-7 cells at moderate hypoxic conditions

TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) is known to promote cancer initiation and progression and accumulates in mammary fat tissue. Effects of TCDD are mediated by the aryl hydrocarbon receptor (AhR). Physiological conditions of moderate hypoxia in breast cancer also activate another transcription factor, hypoxia-inducible factor-1 alpha (HIF-1alpha). In addition, the transcription factors p53 and the estrogen receptor alpha (ERalpha) are important key players in breast cancer progression. Here, human breast cancer cells cultured under mild hypoxic conditions were exposed to TCDD and analyzed for regulation of p53 signaling and ERalpha transactivation. Simultaneous exposure to TCDD and hypoxia resulted in a moderate but reproducible inhibition of p53 expression. Both the direct activation of the ERalpha and the transcriptional regulation of Hdm2 mediated this inhibition. As consequence the p53-mediated target gene expression (Dusp5) was reduced. Silencing of Dusp5 by simultaneous exposure of TCDD and hypoxia or by RNAi led to increased phosphorylation of ERK1/2. This increase resulted in transactivation of ERalpha and induction of ERalpha-mediated transcription of Hdm2 and SOCS3. Specificity of ERalpha-transactivation by ERK1/2 was confirmed by treatment with MAPKK-inhibitor PD98059. The combination of inhibition of functional p53 protein and induction of ERalpha signaling could serve as a model for the operational sequence of TCDD effects to prevent cell death and promote breast tumor progression.

Characterization of the specific role and activation of the High Mobility Group A1 (HMGA1) in colon cancer

The High Mobility Group A1 (HMGA1) proteins are a class of oncogenes whose specific role in colon and other cancers is currently emerging. Our analysis of the HMGA1 promoter region revealed several putative sites for beta‐catenin/tcf binding suggesting that HMGA1 may be activated by the adenomatous polyposis coli (Apc)/beta‐catenin pathway in colon cancer. To better understand the specific role of HMGA1 in colon carcinoma formation, we evaluated the expression of HMGA1 in the Apc/min colon cancer mouse model. Results of quantitative real‐time RT‐PCR (qRT‐PCR) show a 9‐fold increase over normal intestine in HMGA1 mRNA where Apc is truncated and in tumors. Consistent with qRT‐PCR data, HMGA1 protein levels were also significantly higher in Apc‐min intestinal tumors than those from normal mouse tissues. To determine if Apc/beta catenin signaling directly impacts HMGA1 expression, we tested the effect of Apc recovery on HMGA1 expression. Preliminary results indicate that zinc‐induced full‐length Apc expression suppresses HMGA1 levels and that truncations in Apc may activate HMGA1 to promote cancer initiation and progression. Collectively, these results provide the first line of evidence supporting the cooperation of truncated Apc and HMGA1 in colon cancer.This project was supported by NIH grant P20 RR‐16461 in support of SC INBRE.

Declining cellular fitness with age promotes cancer initiation by selecting for adaptive oncogenic mutations

Age is the single most important prognostic factor in the development of many cancers. The major reason for this age-dependence is thought to be the progressive accumulation of oncogenic mutations and epigenetic changes. Similarly, mutagens are thought to be carcinogenic primarily by engendering oncogenic mutations. Yet while the accumulation of heritable somatic changes is expected to augment the incidence of oncogenic mutations, a major effect of increased mutation load is reduced fitness. We propose that the fitness of progenitor cell compartments substantially impacts on the selective advantage conferred by particular mutations. We hypothesize that reduced cellular fitness within aged stem cell pools can select for adaptive oncogenic events and thereby promote the initiation of cancer. Thus, certain oncogenic mutations may be adaptive within aged but not young stem cell pools. We further argue that accumulating genetic alterations with age or mutagen exposure might promote cancer not only by causing oncogenic hits within cells but also by leading to eventual reduction in stem cell fitness, which then selects for oncogenic events. Therefore, initial stages of cancer development may not be limited by the incidence of initiating oncogenic changes, but instead by contexts of reduced cellular fitness that select for these changes.