Gene expression, cellular diversification and tumor progression to the metastatic phenotype.

Alterations in the expression of certain genes or in their products can render benign tumor cells metastatic. Experimentally this has been quickly performed by transferring dominantly acting oncogenes such as c-H-rasEJ into susceptible cells, but in vivo such a rapid qualitative change in a dominantly acting oncogene occurs only rarely, and progression to highly metastatic phenotypes is thought to occur through a slow stepwise process. Such slow changes can be reversible and need not involve known dominantly acting oncogenes, consistent with clinical observations. An important element of the natural progression of tumors to malignancy may be their ability to circumvent microenvironmental controls that regulate growth and cellular diversity and to evolve into heterogeneous phenotypes, a process that appears to involve mainly quantitative changes in gene expression but which can be rapidly stimulated in cell culture by the introduction of a dominantly acting oncogene. It is proposed that the highly malignant cells that have slowly evolved in vivo with only a few qualitative gene changes have undergone extensive cycles of diversification and accumulation of quantitative changes in the expression of genes that encode products that are related to malignancy and metastasis. Thus, highly malignant cellular phenotypes can arise quickly through specific qualitative changes in critical controlling genes or more slowly by less critical qualitative genetic changes, coupled with cellular diversification and accumulation of quantitative changes in gene expression.     

Dominant and recessive genes involved in tumor cell invasion

The past year has been the discovery and further analysis of several genes and protein products that are critically involved in the generation of invasive and metastatic tumor cells. Like oncogenes and tumor suppressor genes, the genes responsible for invasive and metastatic phenotypes can function in a dominant or recessive fashion. In this review, particular emphasis will be given to the dominantly acting genes encoding the cell adhesion molecule CD44 and the motility factor scatter factor, and the recessively acting genes encoding the cell adhesion molecule E-cadherin and nm23.     

Intrinsic or acquired drug resistance and metastasis: Are they linked phenotypes?

Evidence is reviewed which suggests a linkage may exist between certain forms of de novo or acquired drug resistance and metastasis. This includes finding that expression of certain dominantly acting mutant oncogenes or tumor suppressor genes, e.g. genes which normally act to “drive” tumor progression and metastasis, can also affect the expression of drug resistance. Moreover, this can be accompanied by altered expression of certain cellular genes thought to be involved in expression of drug resistance. A direct linkage between acquired drug resistance and metastasis would suggest that tumor sublines selected for drug resistance should manifest more aggressive malignant properties than their drug-sensitive counterparts. While this does not appear to be true for drug resistant sublines selected in vitro, indeed such cell lines frequently manifest diminished in vivo tumorigenic and/or metastatic competence, there is some evidence to support such a correlation exists for tumor cell lines that are selected in vivo for drug resistance. Attention is also drawn to the fact that new linkages between metastasis and drug resistance may be uncovered by analyzing the ability of tumor subpopulations to acquire drug resistance after one or several previous exposures to chemotherapeutic drugs, as opposed to examining intrinsic drug resistance only. Furthermore, ability to detect induced or acquired drug resistance in vitro may be strongly influenced by the types of assay used to detect and monitor drug resistanc. In particular, three-dimensional cell culture systems may reveal acquired or induced “multicellular” drug resistance in situations where conventional two-dimensional culture systems may therefore reveal as yet undiscovered associations between the phenotypes of metastasis and drug resistance.     

Transformation effector and suppressor genes.

Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto-oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes is referred to by a number of different names including; anti-oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.     

Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer.

Carcinogenesis is a multistage process that has been characterized both by the activation of cellular oncogenes and by the loss of function of tumor suppressor genes. Colorectal cancer has been associated with the activation of ras oncogenes and with the deletion of multiple chromosomal regions including chromosomes 5q, 17p, and 18q. Such chromosome loss is often suggestive of the deletion or loss of function of tumor suppressor genes. The candidate tumor suppressor genes from these regions are, respectively, MCC and/or APC, p53, and DCC. In order to further our understanding of the molecular and genetic mechanisms involved in tumor progression and, thereby, of normal cell growth, it is important to determine whether defects in one or more of these loci contribute functionally in the progression to malignancy in colorectal cancer and whether correction of any of these defects restores normal growth control in vitro and in vivo. To address this question, we have utilized the technique of microcell-mediated chromosome transfer to introduce normal human chromosomes 5, 17, and 18 individually into recipient colorectal cancer cells. Additionally, chromosome 15 was introduced into SW480 cells as an irrelevant control chromosome. While the introduction of chromosome 17 into the tumorigenic colorectal cell line SW480 yielded no viable clones, cell lines were established after the introduction of chromosomes 15, 5, and 18. Hybrids containing chromosome 18 are morphologically similar to the parental line, whereas those containing chromosome 5 are morphologically distinct from the parental cell line, being small, polygonal, and tightly packed. SW480-chromosome 5 hybrids are strongly suppressed for tumorigenicity, while SW480-chromosome 18 hybrids produce slowly growing tumors in some of the animals injected. Hybrids containing the introduced chromosome 18 but was significantly reduced in several of the tumor reconstitute cell lines. Introduction of chromosome 5 had little to no effect on responsiveness, whereas transfer ot chromosome 18 restored responsiveness to some degree. Our findings indicate that while multiple defects in tumor suppressor genes seem to be required for progression to the malignant state in colorectal cancer, correction of only a single defect can have significant effects in vivo and/or in vitro.     

The conversion of mouse skin squamous cell carcinomas to spindle cell carcinomas is a recessive event

Squamous carcinomas of both human and rodent origin can undergo a transition to a more invasive, metastatic phenotype involving reorganization of the cytoskeleton, loss of cell adhesion molecules such as E-cadherin and acquisition of a fibroblastoid or spindle cell morphology. We have developed a series of cell lines from mouse skin tumors which represent different stages of carcinogenesis, including benign papillomas, and clonally related squamous and spindle carcinomas derived from the same primary tumor. Some spindle cells continue to express keratins, but with a poorly organized keratin filament network, whereas in others no keratin expression is detectable. All of the spindle cells lack expression of the cell adhesion molecule E-cadherin and the desmosomal component desmoplakin. Loss of these cell surface proteins therefore appears to precede the destabilization of the keratin network. At the genetic level, it is not known whether such changes involve activation of dominantly acting oncogenes or loss of a suppressor function which controls epithelial differentiation. To examine this question, we have carried out a series of fusion experiments between a highly malignant mouse skin spindle cell carcinoma and cell lines derived from premalignant or malignant mouse skin tumors, including both squamous and spindle carcinoma variants. The results show that the spindle cell phenotype as determined by cell morphology and lack of expression of keratin, E-cadherin, and desmoplakin proteins, is recessive in all hybrids with squamous cells. The hybrids expressed all of these differentiation markers, and showed suppression of tumorigenicity to a variable level dependent upon the tumorigenic properties of the less malignant fusion partner. Our results suggest that acquisition of the spindle cell phenotype involves functional loss of a gene(s) which controls epithelial differentiation.     

Oncogenes and anti-oncogenes in tumorigenesis

Recent advances have led to the identification of cellular genes which are involved in the initiation and progression of tumorigenesis. The proto-oncogenes, which normally participate in the regulation of cell proliferation and differentiation, can become oncogenes through alterations in the regulation of their expression and/or their coding sequences. Their contribution to the tumorigenic phenotype is dominant. The anti-oncogenes or tumor suppressor genes or recessive oncogenes are normally implicated in a negative regulation of cellular proliferation. The loss of their activity contributes to tumorigenesis in a recessive manner. Genetic events activating proto-oncogenes or inactivating anti-oncogenes accumulate in the same cell during tumor progression and co-operate to determine the malignant invasive phenotype of advanced tumors.     

Transition to androgen-independence in prostate cancer

Prostate carcinoma is the most frequently diagnosed malignancy and the second leading cause of death as a result of cancer in men in the western countries. Withdrawal of androgens or the peripheral blockage of androgen action remain the critical therapeutic options for the treatment of advanced prostate cancer. However, after initial regression, most of the prostate cancers become androgen-independent and progress further, with eventual fatal outcome. Understanding the mechanisms of transition to androgen independence and tumor progression in prostate cancer is critical to finding new ways to treat aged patients that are ineligible for conventional chemotherapy. A large number of different molecular mechanisms might be responsible for the transition to androgen-independence. Many of these involve the androgen receptor (AR) and its signalling pathways, but they might also include genetic changes that affect several genes, which results in the activation of oncogenes or the inactivation of tumor suppressor genes. Here, we discuss the most recent and relevant findings on androgen resistance in prostate cancer in order provide a comprehensive interpretation of the clinical behaviour of tumors at molecular levels.     

The tumor microenvironment and its contribution to tumor evolution toward metastasis

Cancer cells acquire cell-autonomous capacities to undergo limitless proliferation and survival through the activation of oncogenes and inactivation of tumor suppressor genes. Nevertheless, the formation of a clinically relevant tumor requires support from the surrounding normal stroma, also referred to as the tumor microenvironment. Carcinoma-associated fibroblasts, leukocytes, bone marrow-derived cells, blood and lymphatic vascular endothelial cells present within the tumor microenvironment contribute to tumor progression. Recent evidence indicates that the microenvironment provides essential cues to the maintenance of cancer stem cells/cancer initiating cells and to promote the seeding of cancer cells at metastatic sites. Furthermore, inflammatory cells and immunomodulatory mediators present in the tumor microenvironment polarize host immune response toward specific phenotypes impacting tumor progression. A growing number of studies demonstrate a positive correlation between angiogenesis, carcinoma-associated fibroblasts, and inflammatory infiltrating cells and poor outcome, thereby emphasizing the clinical relevance of the tumor microenvironment to aggressive tumor progression. Thus, the dynamic and reciprocal interactions between tumor cells and cells of the tumor microenvironment orchestrate events critical to tumor evolution toward metastasis, and many cellular and molecular elements of the microenvironment are emerging as attractive targets for therapeutic strategies.     

Qualitative and quantitative changes in nuclear DNA and phenotypic gene expression in human malignant skin tumors during their progression.

Qualitative and quantitative changes in nuclear DNA and phenotypic expression of human malignant skin tumors were examined during the course of progression. The numerical abnormalities of chromosomes demonstrated by interphase cytogenetics using the chromosome-specific in situ hybridization technique, were also used to reveal qualitative DNA changes in malignant tumor cells. For the analysis of the quantitative changes in nuclear DNA, fluorescence cytophotometry was used on the DAPI-stained tumor cells isolated from the paraffin-embedded sections. To survey abnormal gene expression in malignant tumor cells, lectin histochemistry for different sugar residues, immunohistochemical staining of HLA-DR, and in situ hybridization for H-ras, c-myc, N-myc or v-fos were used. The results showed that: 1) in one case of squamous cell carcinoma with invasion, the number of chromosomal abnormalities was much greater in the invasive than in non-invasive parts, with marked topographical heterogeneities; 2) the DNA-ploidies were largely shifted to the higher side with aneuploid stem-lines and polyploid cells in the invasive parts of all malignant tumors; 3) the expression of HLA-DR was induced at the invasive fronts of malignant melanomas; 4) the GS-I specific sugar residue(D-galactose) appeared in all extra-mammary Paget's cells; and 5) expression of "oncogenes" was found in about 60% of all malignant tumors examined. Thus, the progression of malignancy is accompanied by both qualitative and quantitative changes in nuclear DNA, resulting in abnormal gene expression.     

Apoptosis in skin cancer development and regression

Non-melanoma skin cancers (NMSC) are the most common malignant tumors in white population and their incidence has been increasing worldwide. Molecular events regulating cell survival, apoptosis, growth arrest as well as cell differentiation, are important contributors to the overall kinetics of benign and malignant cell growth and play a role in their development, progression and regression. Failure of these pathways can result in the loss of control over proliferation and lead to tumor development through the inactivation of tumor suppressor genes or the activation of oncogenes. Also, immunological mechanisms have been implicated in a phenomenon of tumor progression as well as spontaneous tumor regression. We have tried to summarize the main events in etiopatogenesis, development, progression and in some cases skin cancer regression. Further studies are needed to elucidate completely the details of apoptotic control in normal skin and determine factors resulting in apoptotic disbalance and disease.     

Biological processes, properties and molecular wiring diagrams of candidate low-penetrance breast cancer susceptibility genes

Background

The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples.

Methods

We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes.

Results

We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype.

Conclusion

The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.     

Comprehensive genetic analysis of cancer cells

Human cancer is viewed as a disorder of genes originating from the progeny of a single cell that has accumulated multiple genetic alterations. The genetic alterations include point mutation, chromosomal rearrangements and imbalances. Amplifications primarily involve oncogenes whose overexpression leads to growth deregulation, while deletions commonly target tumor suppressor genes that control cell cycle checkpoints and DNA repair mechanisms. With the advent of molecular cytogenetics procedures for global detection of genomic imbalances and for multicolor visualization of structural chromosome changes, as well as the completion of human genome mapping and the development of microarray technology for serial gene expression analysis of the entire genomes, a significant progress has been made in uncovering the molecular basis of cancer. The major challenge in cancer biology is to decipher the molecular anatomy of various cancers and to identify cancer-related genes that now comprise only a fraction of human genes. The complete genetic anatomy of specific cancers would allow a better understanding of the role of genetic alterations in carcinogenesis, provide diagnostic and prognostic markers and discriminate between cells at different stages of progression toward malignancy. This review highlights current technologies that are available to explore cancer cells and outlines their application to investigations in human hepatocellular carcinoma.     

Suppressor genes for malignant and anchorage-independent phenotypes located on human chromosome 9 have no dosage effects

We have previously shown that microcell-mediated transfer of a der(9)t(X;9) human chromosome (HSA), derived from human fibroblast strain GM0705, into the Syrian hamster cell line BHK-191-5C produced only near-tetraploid hybrids, although the recipient cell line contained a 1:1 ratio of near-diploid and near-tetraploid cells. However, the tumorigenicity and the anchorage independence could be suppressed in the near-tetraploid hybrids with one copy of the der(9)t(X;9) chromosome. The introduction of an HSA X chromosome did not suppress either of these phenotypes. We concluded that in addition to two suppressor genes, one for tumorigenicity and another for anchorage independence, HSA 9 might carry a third gene capable of inhibiting cellular growth in vitro, which had dosage effects. In the present study, keeping one copy of the der(9)t(X;9) chromosome, we have increased the hamster background chromosome number beyond hexaploid level by fusing two microcell-generated hybrid cell lines, where both malignant and anchorage-independent phenotypes were suppressed, with the parental malignant BHK-191-5C cell line. Tests with nude mice showed that hybrids containing one copy of the der(9)t(X;9) chromosome against the increased background of chromosomes of malignant parental origin were still suppressed for both phenotypes. These results suggest that the suppressor genes for malignancy and for anchorage independence have no dosage effects, in contrast to the suppressor gene(s) for cellular growth.     

The organizing principle: microenvironmental influences in the normal and malignant breast

The current paradigm for cancer initiation and progression rests on the groundbreaking discoveries of oncogenes and tumor suppressor genes. This framework has revealed much about the role of genetic alterations in the underlying signaling pathways central to normal cellular function and to tumor progression. However, it is clear that single gene theories or even sequential acquisition of mutations underestimate the nature of the genetic and epigenetic changes in tumors, and do not account for the observation that many cancer susceptibility genes (e.g. BRCA1, APC) show a high degree of tissue specificity in their association with neoplastic transformation. Therefore, the cellular and tissue context itself must confer additional and crucial information necessary for mutated genes to exert their influence. A considerable body of evidence now shows that cell-cell and cell-extracellular matrix (ECM) interactions are essential organizing principles that help define the nature of the tissue context, and play a crucial role in regulating homeostasis and tissue specificity. How this context determines functional integrity, and how its loss can lead to malignancy, appears to have much to do with tissue structure and polarity.     

Linear Model of Colon Cancer Initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Linear model of colon cancer initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

On the path to understanding the nature of cancer

In this essay crucial problems of the origin of cancer and the development of malignancy are discussed. The problem of precancer and three ways leading to malignancy are considered: induction of tumor precursors, accumulation of genetic traits common for tumor growth, and the role of inflammation in tumor induction. The nature of viral oncogenes and modes of their action are described in the context of their origin as a component of the viral genome. Oncogenes of RNA-containing viruses and DNA-containing tumorigenic viruses are described together with cellular protooncogenes, which are progenitors of RNA-containing viral oncogenes. Hematological malignancies are described as an intermediate form between simple tumors induced by a single oncogene and more complicated epithelial tumors. The roles of tumor suppressor genes and the interaction of several oncogenes in the formation of carcinomas and also the role of progression in tumor evolution are discussed.