Progression Of Solid Malignancies Research Articles

Three-dimensional growth reveals fine-tuning of 5-lipoxygenase by proliferative pathways in cancer.

The leukotriene (LT) pathway is positively correlated with the progression of solid malignancies, but the factors that control the expression of 5-lipoxygenase (5-LO), the central enzyme in LT biosynthesis, in tumors are poorly understood. Here, we report that 5-LO along with other members of the LT pathway is up-regulated in multicellular colon tumor spheroids. This up-regulation was inversely correlated with cell proliferation and activation of PI3K/mTORC-2- and MEK-1/ERK-dependent pathways. Furthermore, we found that E2F1 and its target gene MYBL2 were involved in the repression of 5-LO during cell proliferation. Importantly, we found that this PI3K/mTORC-2- and MEK-1/ERK-dependent suppression of 5-LO is also existent in tumor cells from other origins, suggesting that this mechanism is widely applicable to other tumor entities. Our data show that tumor cells fine-tune 5-LO and LT biosynthesis in response to environmental changes repressing the enzyme during proliferation while making use of the enzyme under cell stress conditions, implying that tumor-derived 5-LO plays a role in the manipulation of the tumor stroma to quickly restore cell proliferation.

Macrophages as a Potential Immunotherapeutic Target in Solid Cancers.

The revolution in cancer immunotherapy over the last few decades has resulted in a paradigm shift in the clinical care of cancer. Most of the cancer immunotherapeutic regimens approved so far have relied on modulating the adaptive immune system. In recent years, strategies and approaches targeting the components of innate immunity have become widely recognized for their efficacy in targeting solid cancers. Macrophages are effector cells of the innate immune system, which can play a crucial role in the generation of anti-tumor immunity through their ability to phagocytose cancer cells and present tumor antigens to the cells of adaptive immunity. However, the macrophages that are recruited to the tumor microenvironment predominantly play pro-tumorigenic roles. Several strategies targeting pro-tumorigenic functions and harnessing the anti-tumorigenic properties of macrophages have shown promising results in preclinical studies, and a few of them have also advanced to clinical trials. In this review, we present a comprehensive overview of the pathobiology of TAMs and their role in the progression of solid malignancies. We discuss various mechanisms through which TAMs promote tumor progression, such as inflammation, genomic instability, tumor growth, cancer stem cell formation, angiogenesis, EMT and metastasis, tissue remodeling, and immunosuppression, etc. In addition, we also discuss potential therapeutic strategies for targeting TAMs and explore how macrophages can be used as a tool for next-generation immunotherapy for the treatment of solid malignancies.

Impact of Chromosome 9 Numerical Imbalances in Oral Squamous Cell Carcinoma: A Pilot Grid-Based Centromere Analysis.

Oral squamous cell carcinoma (OSCC) is considered an aggressive malignancy, mainly due to its increased propensity to provide local and distant lymph node metastases. Gross chromosome instability (CI; polysomy/aneuploidy/monosomy), combined or not with specific gene alterations, is implicated in the development and progression of solid malignancies, including OSCC. In order to further study the relationship between these genetic alterations and the aggressive biological behavior of OSCCs, we investigated the frequency and impact of chromosome 9 numerical imbalances in these tumors. Fifty (n = 50) formalin-fixed, paraffin-embedded primary OSCC tissue sections were used. Chromogenic in situ hybridization (CISH) was implemented for detecting chromosome 9 (CEN—centromere enumeration) numerical alterations. Concerning the screening process in CISH slides, a novel, real-time reference and calibration grid platform was implemented. Chromosome 9 polysomy was observed in 8/50 (16%) tissue sections, whereas the rest of them demonstrated a normal, diploid pattern (42/50; 84%). Chromosome 9 polysomy was associated with the grade of differentiation of the examined tumors (p = 0.036). Chromosome 9 numerical imbalances (polysomy) were observed in sub-groups of OSCCs correlating with a progressive dedifferentiation of the malignant tissues. Concerning the implementation of the proposed grid-based platform as described above on CISH slides, it provides a novel, fast, and accurate screening mapping mechanism for detecting chromosome numerical imbalances.

Cyclin D1 Gene Numerical Imbalances in Laryngeal Squamous Cell Carcinoma: A Tissue Microarray Grid Based Analysis.

Background:Deregulation of critical proteins involved in cell cycle stability, such as cyclins, is a frequent genetic event in the development and progression of solid malignancies. Concerning laryngeal squamous cell carcinoma (LSCC), cyclin D1 oncogenic transformation leads to an aberrant protein expression and seems to affect the biological behaviour of the neoplasm. The aim of this study was to determine the correlation of cyclin D1 numerical imbalances with the corresponding protein expression levels in LSCCs. Material and Method:Using tissue microarray (TMA) technology, fifty (n=50) histologically confirmed primary LSSCs were cored at a diameter of 1.5 mm. Immunohistochemistry (IHC) and chromogenic in situ hybridization (CISH) analyses were applied. Concerning the screening process in CISH slides, a novel real-time reference and calibration grid platform was implemented. Results:Protein overexpression was observed in 22/50 (44%) cases; whereas, gene amplification was seen in 13/50 (26%) cases (p=0.02). Combined protein/ gene deregulation was associated with the stage of malignancy (p= 0.0014, p=0.001), whereas overall protein expression was strongly correlated with the grade of tumour (p= 0.001). Conclusion: Cyclin D1 gene amplification led to aberrant protein expression in LSCCs and it was also correlated with an aggressive biological behaviour. To best of our knowledge, this study was the first described grid based CISH analysis under conventional bright field microscopy for detecting gene numerical imbalances while providing a novel and accurate description for screening-mapping process in the entire slide area.

PO-219 New cell genetic tracing and single-cell tools for the study of the molecular pathways involved in cancer metastasis

IntroductionTraditionally, metastasis has been seen as the final and often fatal step in the progression of solid malignancies. This vision of tumour progression has been recently challenged, as genetic analyses of circulating tumour cells and functional studies in animal models, have suggested that the dissemination of tumour cells can be a very early event, even at a premalignant stage. Whether this implies that the colonisation of the distant tissue happens at such an early stage is still unclear. In accordance with an early metastatic implantation model, there is evidence in cancer of early parallel evolution of primary and metastatic tumours as well as tissue specific evolutionary branches among different metastases from the same tumour. Transcriptional analysis of cells with different metastatic potential in mouse tissues have been used to identify the genes and pathways involved in metastasis specificity. Moreover, intratumour heterogeneity has been observed in multiple cancers and has been postulated as a critical aspect for tumour metastasis and treatment resistance. In this context, the use of new molecular and sequencing strategies like cell lineage tracing systems and single-cell sequencing, to genetically modified mouse models, could provide new opportunities to unravel the molecular mechanisms behind metastatic potential.Material and methodsIn order to construct a new fluorescent-based lineage tracing system, we have performed a systematic evaluation of the fluorescent characteristics of different proteins and have tried several orientation and locations of loxP sites in order to provide a random and proportional repertoire of fluorescent labelling after CRE recombination. Additionally, we have set up droplet-based microfluidic technology to perform single-cell sequencing on murine tumour primary samples.Results and discussionsHere we have constructed a new allele able to produce up to 15 different colour combinations that can be uniquely identified by confocal microscopy and FACS. Additionally, we have set up the infrastructure and protocol to perform single-cell RNA-Seq and targetted sequencing on tumour primary samples.ConclusionWe have generated very promising new tools that could open new opportunities to study the molecular mechanisms behind metastatic potential in mouse and human tumours.

Genetic Polymorphisms of Insulin-Like Growth Factor 1 Are Associated with Osteosarcoma Risk and Prognosis.

BackgroundInsulin-like growth factor 1 (IGF-1) gene plays an important role in bone and soft tumors. IGF-1 gene polymorphisms have been revealed to be correlated with the carcinogenesis and progression of solid malignancies. We therefore hypothesized that IGF-1 genetic polymorphisms might be associated with the risks and outcomes of osteosarcomas in Chinese individuals.Material/MethodsThis study included 173 conventional osteosarcoma individuals and 175 tumor-free controls. Five single nucleotide polymorphisms (SNPs) of IGF-1 (rs6214, rs6218, rs35767, rs5742612, and rs5742714) were genotyped. DNA was extracted from peripheral blood and analyzed for SNP genotyping using PCR.ResultsWe found that rs6218 had a predictive role for the susceptibility and progression of osteosarcoma. The presence of TC and CC genotypes of rs6218 indicated higher risk of osteosarcoma. In addition, rs6218 TC and CC genotypes were discovered to be associated with later stage and elevated risk of osteosarcoma metastasis.ConclusionsIGF-1 polymorphisms are potential prognostic predictors of osteosarcoma susceptibility and outcomes.

Abstract 1568: Evaluate the efficacy of new therapeutics using advanced metastatic mouse models.

Abstract Metastasis, the spread and growth of tumor cells to distant organs, is usually a final and fatal step in the progression of solid malignancies. Preclinical test of the new anti-cancer therapies that could mimic the disease present in most clinical trials asks for robust metastatic models than just the subcutaneous models. We sought to establish and validate a panel of experimental (i.v. inoculation) and spontaneous (orthotopic inoculation) mouse models with high metastatic potential and varied tissue tropism in different types of cancer models, covering colorectal, liver, prostate, breast, pancreatic cancer and melanoma. The responsiveness of metastasis to several therapies that are proved to be clinically effective or ineffective was investigated. A higher predictive power of the metastatic models was demonstrated compared with the corresponding subcutaneous models. By means of the transcriptomic analysis of the paired primary tumor population and the distant lung metastasis population in 5 orthotopic tumor models, we identified the gene signatures that differentiate the two populations in different tumor types. Our signature was also compared with the clinical relevant gene signatures correlated with lung metastasis risk. Several genes are concordantly expressed in both signatures and are biologically meaningful. Overall, given the higher predictive power and the clinical relevance, the use of the metastatic mouse models constitutes an effective and important strategy to preclinically evaluate the efficacy of new therapeutic regimens. Citation Format: Juan Zhang, Sheng Guo, Jie Cai, Yanmei Sun, Guanping Mao, Zhun Wang, Lan Zhang, Ying Jin, Qian Shi, Taiping Chen. Evaluate the efficacy of new therapeutics using advanced metastatic mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1568. doi:10.1158/1538-7445.AM2013-1568

Abstract 2810: Evaluating MMP contribution to pre-metastatic niche formation.

Abstract Metastasis, the fatal progression of solid malignancies, consists of a complex and multistep process characterized by a series of ordered biological events. One step in this process occurs when stromal microenvironmental changes take place at secondary sites before the arrival of tumor cells. These modified tissue-specific sites are induced by signals from the primary tumor and further supported by recruitment of progenitor bone marrow derived cells and are commonly referred to as “pre-metastatic niches.” Matrix metalloproteinases (MMPs) are crucially involved in many pathological processes including tumor invasion and metastasis. Our goal is to determine the roles of MMPs in generation of a pre-metastatic niche in different target organs, specifically the lung and liver. Using an MMP proteolytic beacon capable of detecting MMP activity in situ, we observed increased signal in lungs of mice carrying pre-metastatic mammary tumors. In a LLC-implantation model, qPCR was used to profile MMP expression in pre-metastatic tissues versus controls and indicated that in the lung, MMP9 (gelatinase B) was increased. Further, we have developed staining and quantitation techniques to define pre-metastatic niches. These were also based on molecules identified by Kaplan et al such as VEGFR-1, VLA-4 and fibronectin. Immunofluorescent analysis of mouse lungs subcutaneously implanted with LLC cells demonstrated clusters of cells that positively co-stained for VEGFR-1, VLA-4 and Fibronectin. We also tested whether soluble factors from metastatic cell lines could induce changes in lungs of mice. Analysis of MMP-9 levels as well as localization of the known markers both failed to show any significant changes in the lung, leading us to believe that the presence of a primary tumor in vivo is required for pre-metastatic niche formation. We are now working with the well-characterized metastatic mammary tumor line 4T1 to test whether the changes we have seen with LLC cells are reproducible in a different cancer type and mouse strain. Additionally we are testing whether the absence of host MMP-9 affects the formation of a pre-metastatic niche in either model. The increased presence of MMP-9 and mentioned factors before tumor cell arrival could indicate potential roles and markers for metastatic spread, possibly leading to potential therapeutics for preventing metastasis and increasing overall cancer survival. Egeblad et al. Journal of Biochemistry and Molecular Biology, Vol. 36, No. 1, January 2002, pp. 128-137 Kaplan et el. Nature, Vol. 438, No. 7069, December 2005, pp 820-827 Citation Format: Elizabeth A. Dozier, J. Oliver McIntyre, Conor Lynch, Lynn Matrisian, Barbara Fingleton. Evaluating MMP contribution to pre-metastatic niche formation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2810. doi:10.1158/1538-7445.AM2013-2810

The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition

BackgroundMuch attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables.ResultsOur data indicate that human prostate cancer cell lines contain a small population of CD44+CD133+ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44+α2β1+CD133+ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSC's migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSC's migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.ConclusionSince carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment.

CD133 antigen expression in ovarian cancer

BackgroundMuch attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively, thus sustaining tumor growth, the identification of CSCs through their antigenic profile might have relevant clinical implications. In this context, CD133 antigen has proved to be a marker of tumor cells with stemness features in several human malignancies.The aim of the study was to investigate the clinical role of the immunohistochemically assessed expression of CD133 in a large single Institution series of ovarian cancer patients.MethodsThe study included 160 cases admitted to the Gynecologic Oncology Unit, Catholic University of Campobasso and Rome. CD133 antigen was identified by the monoclonal mouse anti-CD133-1 antibody (clone CD133 Miltenyi biotec).ResultsIn the overall series CD133 positive tumor cells were observed in 50/160 (31.2%) cases. A diffuse cytoplasmic pattern was identified in 30/50 (60.0%), while an apical cytoplasmic pattern was found in 20/50 (40.0%) of CD133 positive tumors.As of September 2008, the median follow up was 37 months (range: 2–112). During the follow up period, progression and death of disease were observed in 123 (76.9%), and 88 (55.0%) cases, respectively. There was no difference in TTP between cases with negative (median TTP = 23 months) versus positive CD133 expression (median TTP = 24 months) (p value = 0.3). Similar results were obtained for OS. When considering the TTP and OS curves according to the pattern of CD133 expression, a trend to a worse prognosis for cases with diffuse cytoplasmic versus the apical cytoplasmic pattern was documented, although the statistical significance was not reached.ConclusionThe immunohistochemical assessment of CD133 expression seems not to provide additional prognostic information in ovarian cancer patients. The role of the different pattern of CD133 immunoreaction deserves further investigation in a larger series.