MYC and PIM2 co-expression in mouse bone marrow cells readily establishes permanent myeloid cell lines that can induce lethal myeloid sarcoma in vivo

The hematopoietic cell malignancy is one of the most prevalent type of cancer and the disease has multiple pathologic molecular signatures. Research on the origin of hematopoietic cancer stem cells and the mode of subsequent maintenance and differentiation needs robust animal models that can reproduce the transformation and differentiation event in vivo. Here, we show that co-transduction of MYC and PIM2 proto-oncogenes into mouse bone marrow cells readily establishes permanent cell lines that can induce lethal myeloid sarcoma in vivo. Unlike the previous doubly transgenic mouse model in which coexpression of MYC and PIM2 transgenes exclusively induced B cell lymphoma, we were able to show that the same combination of genes can also transform primary bone marrow myeloid cells in vitro resulting in permanent cell lines which induce myeloid sarcoma upon in vivo transplantation. By inducing cancerous transformation of fresh bone marrow cells in a controlled environment, the model we established will be useful for detailed study of the molecular events involved in initial transformation process of primary myeloid bone marrow cells and provides a model that can give insight to the molecular pathologic characteristics of human myeloid sarcoma, a rare presentation of solid tumors of undifferentiated myeloid blast cells associated with various types of myeloid leukemia.     

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. ¹, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.     

The use of genetically engineered mouse models of prostate cancer for nutrition and cancer chemoprevention research

The ability to modify the expression of specific genes in the mouse through genetic engineering technologies allows for the generation of previously unavailable models for prostate cancer prevention research. Although animal models have existed for some time for the study of prostate cancer prevention (primarily in the rat), it is uncertain if the mechanisms that drive prostate carcinogenesis in these models are relevant to those in human prostate cancer. Cell culture studies are of limited usefulness because the conditions are inherently artificial. Factors such as relevant physiologic concentrations and metabolism of putative chemoprevention compounds are difficult to model in an in vitro system. These studies also preclude the types of interactions known to occur between multiple cell types in vivo. In addition, all prostate cancer cell lines are already highly progressed and are not representative of the type of cells to which most preventive strategies would be targeted. Due to the advent of genetically engineered mouse (GEM) models, we now have models of prostate cancer that are dependent on molecular mechanisms already implicated in human prostate carcinogenesis. With these models we can perform a variety of experiments that could previously only be done in cell culture or in prostate cancer cell line xenografts. The currently available GEM models of prostate cancer have been extensively reviewed therefore, this review will focus on the types of models available and their usefulness for various types of preclinical studies relevant to prostate cancer prevention.     

Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia

Refined cancer models are required if researchers are to assess the burgeoning number of potential targets for cancer therapeutics in a clinically relevant context that allows a fast turnaround. Here we use tumor-associated genetic pathways to transform primary human epithelial cells from the epidermis, oropharynx, esophagus and cervix into genetically defined tumors in a human three-dimensional (3D) tissue environment that incorporates cell-populated stroma and intact basement membrane. These engineered organotypic tissues recapitulated natural features of tumor progression, including epithelial invasion through basement membrane, a complex process that is necessary for biological malignancy in 90% of human cancers. Invasion was rapid and was potentiated by stromal cells. Oncogenic signals in 3D tissue, but not 2D culture, resembled gene expression profiles from spontaneous human cancers. We screened 3D organotypic neoplasia with well-characterized signaling pathway inhibitors to distill a clinically faithful cancer gene signature. Multitissue 3D human tissue cancer models may provide an efficient and relevant complement to current approaches to characterizing cancer progression.     

Functional genetics and experimental models of human cancer

Abundant evidence supports the hypothesis that cancer arises from normal cells through the stepwise accumulation of genetic mutations. The study of cells obtained from patients with cancer has identified numerous molecules and pathways that fundamentally contribute to malignant transformation; however, cancer cell lines are often difficult to isolate or maintain, and the cell lines that are available for experimentation represent only a small subset of late-stage human cancers. Recent work has elucidated the role of telomerase in regulating human cell lifespan and has enabled the development of new experimental systems to study human cancer. This review highlights the recent progress in combining genetic methods and primary human cells to understand the role of specific genes and pathways in cancer pathogenesis.     

Development and Histopathological Characterization of Tumorgraft Models of Pancreatic Ductal Adenocarcinoma

Pancreatic cancer is the one of the deadliest of all malignancies. The five year survival rate for patients with this disease is 3-5%. Thus, there is a compelling need for novel therapeutic strategies to improve the clinical outcome for patients with pancreatic cancer.  Several groups have demonstrated for other types of solid tumors that early passage human tumor xenograft models can be used to define some genetic and molecular characteristics of specific human tumors. Published studies also suggest that murine tumorgraft models (early passage xenografts derived from direct implantation of primary tumor specimens) may be useful in identifying compounds with efficacy against specific tumor types.  Because pancreatic cancer is a fatal disease and few well-characterized model systems are available for translational research, we developed and characterized a panel of pancreatic tumorgraft models for biological evaluation and therapeutic drug testing.  Of the 41 primary tumor specimens implanted subcutaneously into mice, 35 produced viable tumorgraft models.  We document the fidelity of histological and morphological characteristics and of KRAS mutation status among primary (F0), F1, and F2 tumors for the twenty models that have progressed to the F3 generation.  Importantly, our procedures produced a take rate of 85%, higher than any reported in the literature. Primary tumor specimens that failed to produce tumorgrafts were those that either contained <10% tumor cells or that were obtained from significantly smaller primary tumors. In view of the fidelity of characteristics of primary tumor specimens through at least the F2 generation in mice, we propose that these tumorgraft models represent a useful tool for identifying critical characteristics of pancreatic tumors and for evaluating potential therapies.      

Stemness markers characterize IGR-CaP1, a new cell line derived from primary epithelial prostate cancer

Deciphering molecular pathways involved in the early steps of prostate oncogenesis requires both in vitro and in vivo models derived from human primary tumors. However the few recognized models of human prostate epithelial cancer originate from metastases. To date, very few models are proposed from primary tumors and immortalizing normal human prostate cells does not recapitulate the natural history of the disease. By culturing human prostate primary tumor cells onto human epithelial extra-cellular matrix, we successfully selected a new prostate cancer cell line, IGR-CaP1, and clonally-derived subclones. IGR-CaP1 cells, that harbor a tetraploid karyotype, high telomerase activity and mutated TP53, rapidly induced subcutaneous xenografts in nude mice. Furthermore, IGR-CaP1 cell lines, all exhibiting negativity for the androgen receptor and PSA, express the specific prostate markers alpha-methylacyl-CoA racemase and a low level of the prostate-specific membrane antigen PSMA, along with the prostate basal epithelial markers CK5 and CK14. More importantly, these clones express high CD44, CD133, and CXCR4 levels associated with high expression of α2β1-integrin and Oct4 which are reported to be prostate cancer stemness markers. RT-PCR data also revealed high activation of the Sonic Hedgehog signalling pathway in these cells. Additionally, the IGR-CaP1 cells possess a 3D sphere-forming ability and a renewal capacity by maintaining their CSC potential after xenografting in mice. As a result, the hormone-independent IGR-CaP1 cellular clones exhibit the original features of both basal prostate tissue and cancer stemness. Tumorigenic IGR-CaP1 clones constitute invaluable human models for studying prostate cancer progression and drug assessment in vitro as well as in animals specifically for developing new therapeutic approaches targeting prostate cancer stem cells.     

Understanding transformation: progress and gaps

Cancer is a collection of complex genetic diseases characterized by multiple defects in the homeostatic mechanisms that regulate cell growth, proliferation and differentiation. Although the analysis of human tumor specimens has allowed the identification of many molecules and pathways important for the malignant phenotype, we still lack a complete understanding of the events that conspire to program any specific type of cancer. Recent advances in developing human experimental models of cancer have provided new insights into the pathways whose perturbation is necessary to achieve cell transformation. These studies indicate that many combinations of genetic mutations confer tumorigenicity on human cells and that both cell-type and tumor-stromal interactions play critical roles in dictating the tumor phenotype.     

Reprogramming of human cancer cells to pluripotency for models of cancer progression

The ability to study live cells as they progress through the stages of cancer provides the opportunity to discover dynamic networks underlying pathology, markers of early stages, and ways to assess therapeutics. Genetically engineered animal models of cancer, where it is possible to study the consequences of temporal‐specific induction of oncogenes or deletion of tumor suppressors, have yielded major insights into cancer progression. Yet differences exist between animal and human cancers, such as in markers of progression and response to therapeutics. Thus, there is a need for human cell models of cancer progression. Most human cell models of cancer are based on tumor cell lines and xenografts of primary tumor cells that resemble the advanced tumor state, from which the cells were derived, and thus do not recapitulate disease progression. Yet a subset of cancer types have been reprogrammed to pluripotency or near‐pluripotency by blastocyst injection, by somatic cell nuclear transfer and by induced pluripotent stem cell (iPS) technology. The reprogrammed cancer cells show that pluripotency can transiently dominate over the cancer phenotype. Diverse studies show that reprogrammed cancer cells can, in some cases, exhibit early‐stage phenotypes reflective of only partial expression of the cancer genome. In one case, reprogrammed human pancreatic cancer cells have been shown to recapitulate stages of cancer progression, from early to late stages, thus providing a model for studying pancreatic cancer development in human cells where previously such could only be discerned from mouse models. We discuss these findings, the challenges in developing such models and their current limitations, and ways that iPS reprogramming may be enhanced to develop human cell models of cancer progression.     

Crystallization of human c-H-ras oncogene products

There is compelling evidence that cancer develops as a consequence of genetic changes (probably multiple) in some members of a selected set of cellular genes. DNA isolated from a variety of tumors, but not normal tissues, possesses the ability to malignantly transform non-tumorigenic cells. Many oncogenes responsible for such transformation have been isolated from transformed cell lines and animal and human tumors induced spontaneously, by virus, by chemical, or by radiation. The most commonly found transforming genes isolated from human tumor cells by DNA transfection assay are the ras gene family (c-H-ras, c-K-ras and N-ras). We report crystallization of several human c-H-ras oncogene proteins.     

The Importance of Genome Architecture in Cancer Susceptibility: Location,Location, Location

Tumorigenesis requires the interaction between different gene disruptions to convert anormal cell into a cancer cell. These gene disruptions can involve loss of expression ormisexpression of genes through genetic or epigenetic mutations. It is becoming clear that thesedisruptions are not isolated events in the genome, but are affected by genome architecture andthe syntenic relationship of alleles on chromosomes. A better understanding of the genetic andepigenetic changes in cancer is important for the rational design of new therapies. We haverecently shown that background-specific polymorphisms and loci under epigenetic regulationhave a strong effect on cancer susceptibility in a mouse model of astrocytoma. Although thesemice carry mutations in p53 and ras signaling pathways (through mutation of the rasGAPprotein, Nf1), the susceptibility to different tumor types depends strongly on epigeneticregulation and does not show simple Mendelian inheritance. Our results demonstrate theimportance of genome architecture and how tumorigenesis can be accelerated by concomitantloss or gain of multiple genes in a single chromosome rearrangement. Because genomearchitecture is very different between mice and humans, comparing patterns of genomicrearrangement in human cancer and mouse models may help distinguish causal genomic changesfrom correlative changes.     

The role of focal adhesion kinase in tumor initiation and progression

Focal adhesion kinase (FAK) is a nonreceptor tyrosine kinase that acts as a primary regulator of focal adhesion signaling to regulate cell proliferation, survival, and migration. While FAK is known to directly influence many fundamental adhesion and growth factor signaling pathways important in cancer, and FAK is overexpressed in multiple human cancers, studies addressing a causal role for FAK in tumor initiation and progression using transgenic models of human cancer had not been performed. Recently, using tissue-specific FAK-knockout in mouse models of human cancer, the consequences of FAK ablation in carcinoma were demonstrated by multiple independent research groups. Strong consensus evidence indicates that epithelial cells are able to transform in the absence of FAK, but do not undergo a malignant conversion to invasive carcinoma, and as such, metastasis is impaired. This is likely the consequence of decreased Src and p130Cas activation in concert with misregulated actin cytoskeleton dynamics and Rho GTPase signaling. Hence, FAK, as well as the FAK-regulating/regulated signaling network, are viable candidates for cancer metastasis therapies.     

Brain stem cells as the cell of origin in glioma

Glioma incidence rates in the United States are near 20000 new cases per year, with a median survival time of 14.6 mo for high-grade gliomas due to limited therapeutic options. The origins of these tumors and their many subtypes remain a matter of investigation. Evidence from mouse models of glioma and human clinical data have provided clues about the cell types and initiating oncogenic mutations that drive gliomagenesis, a topic we review here. There has been mixed evidence as to whether or not the cells of origin are neural stem cells, progenitor cells or differentiated progeny. Many of the existing murine models target cell populations defined by lineage-specific promoters or employ lineagetracing methods to track the potential cells of origin. Our ability to target specific cell populations will likely increase concurrently with the knowledge gleaned from an understanding of neurogenesis in the adult brain. The cell of origin is one variable in tumorigenesis, as oncogenes or tumor suppressor genes may differentially transform the neuroglial cell types. Knowledge of key driver mutations and susceptible cell types will allow us to understand cancer biology from a developmental standpoint and enable early interventional strategies and biomarker discovery.     

Brain stem cells as the cell of origin in glioma简

Glioma incidence rates in the United States are near 20000 new cases per year, with a median survival time of 14.6 mo for high-grade gliomas due to limited therapeutic options. The origins of these tumors and their many subtypes remain a matter of investigation. Evidence from mouse models of glioma and human clinical data have provided clues about the cell types and initiating oncogenic mutations that drive gliomagenesis, a topic we review here. There has been mixed evidence as to whether or not the cells of origin are neural stem cells, progenitor cells or differentiated progeny. Many of the existing murine models target cell populations defined by lineage-specific promoters or employ lineage-tracing methods to track the potential cells of origin. Our ability to target specific cell populations will likely increase concurrently with the knowledge gleaned from an understanding of neurogenesis in the adult brain. The cell of origin is one variable in tumorigenesis, as oncogenes or tumor suppressor genes may differentially transform the neuroglial cell types. Knowledge of key driver mutations and susceptible cell types will allow us to understand cancer biology from a developmental standpoint and enable early interventional strategies and biomarker discovery.     

Accumulation of genetic alterations and their significance in each primary human cancer and cell line

Analyses of multiple genetic alterations accumulated in each cancer cell is expected to provide useful information to elucidate the molecular mechanisms involved in tumorigenesis. Here, we summarized the results of studies on aberrations of oncogenes and tumor suppressor genes by ourselves and other groups. DNAs analyzed were from particular sets of surgical specimens from human tumors and cancer cell lines derived from non-small cell lung cancers, pancreatic cancers, hepatocellular carcinomas and gliomas. Tumors could be grouped into two types based on the genetic alterations detected. Tumors in group 1 had mutations in genes encoding proteins involved in a limited number of signal transduction cascades such as p16-cyclin D1/CDK4-RB or MDM2-p53-p21, where the aberration of one component seems to be sufficient to cause dysfunction of the cascade. Group 2 contained a subset of tumors in which no alteration was detected in the genes analyzed, even in the advanced stage or established cancer cells, indicating the involvement of completely different oncogenic pathways.     

The role of PML ubiquitination in human malignancies

Tumor suppressors are frequently downregulated in human cancers and understanding of the mechanisms through which tumor cells restrict the expression of tumor suppressors is important for the prognosis and intervention of diseases. The promyelocytic leukemia (PML) protein plays a critical role in multiple tumor suppressive functions, such as growth inhibition, apoptosis, replicative senescence, suppression of oncogenic transformation, and inhibition of migration and angiogenesis. These tumor suppression functions are recapitulated in several mouse models. The expression of PML protein is frequently downregulated in diverse types of human tumors and this downregulation often correlates with tumor progression. Recent evidence has emerged that PML is aberrantly degraded in various types of tumors through ubiquitination-dependent mechanisms. Here, we summarize our current understanding of the PML ubiquitination/degradation pathways in human cancers. We point out that multiple pathways lead to PML ubiquitination and degradation. Furthermore, the PML ubiquitination processes are often dependent on other types of posttranslational modifications, such as phosphorylation, prolylisomerization, and sumoylation. Such feature indicates a highly regulated nature of PML ubiquitination in different cellular conditions and cell contexts, thus providing many avenues of opportunity to intervene PML ubiquitination pathways. We discuss the potential of targeting PML ubiquitination pathways for anti-cancer therapeutic strategies.     

Identification and targeting of cancer stem cells

Cancer stem cells (CSC) represent malignant cell subsets in hierarchically organized tumors, which are selectively capable of tumor initiation and self‐renewal and give rise to bulk populations of non‐tumorigenic cancer cell progeny through differentiation. Robust evidence for the existence of prospectively identifiable CSC among cancer bulk populations has been generated using marker‐specific genetic lineage tracking of molecularly defined cancer subpopulations in competitive tumor development models. Moreover, novel mechanisms and relationships have been discovered that link CSC to cancer therapeutic resistance and clinical tumor progression. Importantly, proof‐of‐principle for the potential therapeutic utility of the CSC concept has recently been provided by demonstrating that selective killing of CSC through a prospective molecular marker can inhibit tumor growth. Herein, we review these novel and translationally relevant research developments and discuss potential strategies for CSC‐targeted therapy in the context of resistance mechanisms and molecular pathways preferentially operative in CSC.     

Stem cells in the etiology and treatment of cancer

Using approaches first applied in human leukemias, recent progress has been made in the identification of putative cancer stem cells in several different carcinomas and other solid cancers. Additional studies have suggested that cancer stem cells may be derived not only from transformation of quiescent, long-term stem cells but also from short-lived progenitors that then obtain the ability to undergo self-renewal. Therefore, the heterogeneity observed in many types of human cancers may reflect the activation of specific oncogenes and/or loss of specific tumor suppressor genes and the different stem and/or progenitor cell populations in which these genetic or epigenetic events occur. Similarities have been observed in the pathways regulating stem cell homing and metastasis, and increasing evidence also suggests that treatment failure and the recurrence of human cancer may reflect the intrinsic quiescence and drug resistance of cancer stem cells.