Stress-Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas.

Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.

Update on clinical and mechanistic aspects of paraneoplastic syndromes.

Many patients with cancer are afflicted with an array of severe ailments caused by remote effects of the neoplasm on distant organs, which it has not invaded or colonized. The growing local tumor dominates attention, but invisible chemical and cellular components released by this parasitic neoplastic growth can operate below the threshold of detection to derange feedback loops coordinating essential physiological functions. Ultimately, these changes cause serious signs and symptoms, collectively described as paraneoplastic syndromes (PNSs), which significantly diminish the patient's quality of life. PNS can appear before, or after, detection of the cancer and are sometimes surprising or even bizarre. The patterns of clinical changes seen in these patients are caused by a range of mechanisms that reveal intricate networks of communications between different body systems, normally utilized for healthy function. For example, these disorders demonstrate that (1) hormones, peptides, and other long- and short-range signaling molecules produced by the tumor and (2) immune reactions to tumor-related antigens, can mediate diverse destabilizing effects. However, comparative analysis of numerous PNS reveals valuable information indicating that the primary pathogenetic events instigating these disturbances are much more fundamental. This article provides an overview of the diverse clinical manifestations of paraneoplastic disorders, with representative examples and presents evidence that inappropriate gene expression in the tumor, caused by loss of regulatory control, is a novel unifying explanation for such wide effects of the neoplasm on the host. It also discusses treatment options and issues relating to conducting randomized clinical trials on these disorders.

Loss of acinar cell IKKα triggers spontaneous pancreatitis in mice.

Chronic pancreatitis is an inflammatory disease that causes progressive destruction of pancreatic acinar cells and, ultimately, loss of pancreatic function. We investigated the role of IκB kinase α (IKKα) in pancreatic homeostasis. Pancreas-specific ablation of IKKα (Ikkα(Δpan)) caused spontaneous and progressive acinar cell vacuolization and death, interstitial fibrosis, inflammation, and circulatory release of pancreatic enzymes, clinical signs resembling those of human chronic pancreatitis. Loss of pancreatic IKKα causes defective autophagic protein degradation, leading to accumulation of p62-mediated protein aggregates and enhanced oxidative and ER stress in acinar cells, but none of these effects is related to NF-κB. Pancreas-specific p62 ablation prevented ER and oxidative stresses and attenuated pancreatitis in Ikkα(Δpan) mice, suggesting that cellular stress induced by p62 aggregates promotes development of pancreatitis. Importantly, downregulation of IKKα and accumulation of p62 aggregates were also observed in chronic human pancreatitis. Our studies demonstrate that IKKα, which may control autophagic protein degradation through its interaction with ATG16L2, plays a critical role in maintaining pancreatic acinar cell homeostasis, whose dysregulation promotes pancreatitis through p62 aggregate accumulation.

Role of the host stroma in cancer and its therapeutic significance.

Current cancer research focuses mainly upon the cancer cells in malignant tumours and is providing a growing database about aberrations in their genetic composition. However, tumours also contain non-cancerous host tissue, referred to as the stroma, which plays an active and indispensable role in tumour growth and influences the virulence of the neoplasm towards the host. Many cell types inhabit the stroma, amidst apparently inert fibrous and viscous matrix material, composed of complex polysaccharides, proteins and other molecules. Actually, all of these elements are in constant turnover, causing unpredictable evolution in the properties of the community. This article provides pathologic observations and data on reciprocal interactions between these stromal and neoplastic components of tumours and how they change during the course of the disease. Malignant progression depends upon dauntingly intricate communications between different specialised lineages within the cellular society, which enable rapid adaptation to changing circumstances. Opportunistic misuse of such communication networks enables tumour cells to recruit and incorporate adjacent normal stroma into their midst, so that they may grow, infiltrate and parasitise the host. The absolute dependency of primary tumours and metastases on their diverse stromal components for survival and their insatiable need to continuously recruit more stroma to support expansion, renders them vulnerable to strategies capable of disrupting the cellular interactions involved. This dependency is of critical importance for cancer therapy research, and proposed methods for turning this parasitic behaviour of tumours against themselves are suggested below.

Inhibition of focal adhesion kinase (FAK) activity prevents anchorage-independent ovarian carcinoma cell growth and tumor progression.

Recurrence and spread of ovarian cancer is the 5th leading cause of death for women in the United States. Focal adhesion kinase (FAK) is a cytoplasmic protein-tyrosine kinase located on chromosome 8q24.3 (gene is Ptk2), a site commonly amplified in serous ovarian cancer. Elevated FAK mRNA levels in serous ovarian carcinoma are associated with decreased (logrank P = 0.0007, hazard ratio 1.43) patient overall survival, but how FAK functions in tumor progression remains undefined. We have isolated aggressive ovarian carcinoma cells termed ID8-IP after intraperitoneal (IP) growth of murine ID8 cells in C57Bl6 mice. Upon orthotopic implantation within the peri-ovarian bursa space, ID8-IP cells exhibit greater tumor growth, local and distant metastasis, and elevated numbers of ascites-associated cells compared to parental ID8 cells. ID8-IP cells exhibit enhanced growth under non-adherent conditions with elevated FAK and c-Src tyrosine kinase activation compared to parental ID8 cells. In vitro, the small molecule FAK inhibitor (Pfizer, PF562,271, PF-271) at 0.1 uM selectively prevented anchorage-independent ID8-IP cell growth with the inhibition of FAK tyrosine (Y)397 but not c-Src Y416 phosphorylation. Oral PF-271 administration (30 mg/kg, twice daily) blocked FAK but not c-Src tyrosine phosphorylation in ID8-IP tumors. This was associated with decreased tumor size, prevention of peritoneal metastasis, reduced tumor-associated endothelial cell number, and increased tumor cell-associated apoptosis. FAK knockdown and re-expression assays showed that FAK activity selectively promoted anchorage-independent ID8-IP cell survival. These results support the continued evaluation of FAK inhibitors as a promising clinical treatment for ovarian cancer.

Tailored immunoconjugate therapy depending on a quantity of tumor stroma.

The purpose of this study was to clarify the appropriate combination of targeting antibody and conjugate-design of anti-tumor immunoconjugate depending on a quantity of tumor stroma. Most human solid tumors including pancreatic cancer (PC) forming hypovascular and stroma-rich tumor hinders the penetration of monoclonal antibodies (mAbs) into the cells, and that leads to failure of the conventional cell-targeting immunoconjugate strategy. To overcome this drawback, SN-38 as topoisomerase 1 inhibitor was conjugated to a mAb to collagen 4, a plentiful component of the tumor stroma via ester-bond. The immunoconjugate, which was able to release SN-38 in physiological condition outside the cells, was effective to stroma-rich PC-tumor. On the other hand, anti-CD 20 mAb-PEG-SN-38 via carbamate-bond as conventional immunoconjugate, enabled SN-38 to be released by a carboxylesterase inside of the tumor cell following the internalization, showed strong anti-tumor activity against malignant lymphoma as hypervascular and stroma-poor tumor. The conjugate-design, in parallel with the choice of targeting antibodies, should be selected to maximize the therapeutic effect in each individual tumor having a distinct stromal structure.

Clinical and biological implications of the tumor microenvironment.

In normal tissues and organs, the activities of the constituent cells are strictly restricted to the tasks assigned to them during development. In addition they (with the exception of leukocytes) remain inflexibly confined to their territorial domains by regulatory interactions with their neighbors.This creates specialized local micro-environments in which structure and function are orderly, stable and tightly controlled by feed-back loops, within interacting regulatory networks.This system has considerable ability to adapt to changing conditions. In contrast, the microenvironment in regions where tumors are forming and expanding is characterized by progressive loss of specialized or differentiated cellular functions,disorderly molecular signals, and degeneration of microscopical organ structure. This, coupled with the traffic of cells into and out of the tumor, often culminates in local invasion and metastasis to other organs. The nature of these disturbed molecular and cellular interactions is, by definition,highly unstable and increasingly unpredictable as time passes.It also varies between different tumors, sometimes even leading to regression. However, systematic analysis of this dysfunction in the tumor microcosm, using multiple modern research techniques, has revealed that all actively growing primary and secondary neoplasms share an absolute dependency upon support from adjacent non-neoplastic cells of the host. This support, in turn, continuously depends upon dynamic interplay between tumor and host cell populations, via signaling molecules and surface receptors in the tumor microenvironment.Such interplay determines the fate of the growing neoplasm. Such information, described and evaluated in this article, provides important new insights into the etiology of carcinogenesis and how tumor growth, invasion and metastasis might be therapeutically arrested. The facts and concepts assembled below, regarding the cancer microenvironment, demonstrate how modern molecular findings reveal the impact of the wide range of cancer diseases upon the internal cellular, tissue and organ environments of the whole individual and how this applies to designing new work to improve human cancer diagnosis and treatment. The article discusses several specific types of experimentally-induced and clinically common cancers to derive principles useful for interpreting events in the tumor microenvironment,which apply to cancers in general and especially to human malignant disease.

Inappropriate gene expression in human cancer and its far-reaching biological and clinical significance.

This article provides a broad overview of the field of inappropriate gene expression in many organisms across the animal and plant kingdoms as well as its importance to human disease in general and cancer in particular. Study of the topic is especially important for understanding how the chaotic maelstrom of evolving and cascading regulatory genetic interactions in an advancing cancer produces its clinical effects and for designing pragmatic solutions to how such disorder might eventually be tamed. It is emphasized that the topic warrants much more attention in research and in clinical practice because of the added value it brings to refining cancer diagnosis and treatment and to the assessment of prognostic markers. It is also particularly relevant to understanding the etiology and extensive clinical manifestations of paraneoplastic syndromes affecting multiple organs remote from the tumor and the treatment or amelioration of the substantial morbidity that they cause in cancer patients. More broadly, inappropriate expression can be caused by a number of mechanisms including mutations, rearrangements of the genome, and viral insertions and, under prolonged selection pressures, also has the potential to be an agent of evolutionary change.

Cancer-stroma targeting therapy by cytotoxic immunoconjugate bound to the collagen 4 network in the tumor tissue.

Some cytotoxic immunoconjugates have been approved for malignant lymphoma, a representative of hypervascular and stroma-poor tumors. However, many human solid tumors possess abundant intercellular stromata that prevent diffusion of cancer cell-specific monoclonal antibodies (mAb) and become a barrier preventing immunoconjugates from directly attacking cancer cells. Here we show the successful development of a new strategy that overcomes this drawback and achieves a highly localized concentration of a topoisomerase I inhibitor, SN 38, by conjugating it via an ester bond to a mAb targeted against collagen 4, a plentiful component of the tumor stroma. Poly(ethylene glycol) (PEG) was utilized as a spacer, close to each bond, to maintain stability in the blood. Immunoconjugates selectively extravasated from leaky tumor vessels and minimally from normal vessels because the immunoconjugates are too large to pass through normal vessel walls. Stroma-targeting immunoconjugates bound to the stroma to create a scaffold, from which sustained release of cytotoxic agent occurred and the agent subsequently diffused throughout the tumor tissue to damage both tumor cells and vessels. Cancer-stroma-targeting immunoconjugate therapy was thus validated as a new modality of oncological therapy, especially for refractory, stromal-rich cancers.

Cell and tissue interactions in carcinogenesis and metastasis and their clinical significance.

This review describes a new vision for future directions in the study of metastatic cancer biology and pathology. It is based upon clinical and experimental observations on the constituent cell lineages within a neoplasm and on tumour-host interactions. The vision incorporates information from studies in population biology, developmental biology and experimental pathology as well as investigations upon human malignant disease. The assembled information reveals that invasion and metastasis are supra-cellular manifestations of "emergent behavior" among combinations of normal and malignant cell lineages in vivo. Emergent behavior is a combinatorial interactive process in which a population displays new traits which cannot be achieved by individuals acting separately and which subside when the specific population mix disaggregates. Disruption of such pathological interactions in the field of a developing primary or secondary tumour is, therefore, required to disable the malignant population and arrest progression without tissue destruction. These conclusions originate, in part, from principles which govern the sociobiology and group behavior of bees, ants, fish, birds and human societies. In all these social organisms, external factors can disrupt signaling mechanisms and induce expanding self-perpetuating rogue behavior, leading to social disintegration. These principles also apply to cellular societies composing higher animals, which likewise need intrinsic rules to maintain social order and avoid anarchy, and recognition of this is essential for advancing future research on the mechanisms involved in carcinogenesis and metastasis. Summarised evidence is presented here to support the conclusion that miscommunications between cells and tissues in the region of the developing tumour and its metastases are the main direct perpetrators of malignant disease. Genetic lesions (mutations, deletions, translocations, reduplications, etc.), commonly seen in cancers, can significantly disrupt important molecular pathways in the networks of communications needed to sustain orderly tissue/organ structure and function. However, genetic lesions can also, themselves, be induced by abnormal cell interactions initiated by extrinsic carcinogenic agents such as chemicals, viruses, hormones and radiation. The evidence shows that, irrespective of the initiating cause, it is this miscommunication in the region of a developing tumour and its metastases that is ultimately responsible for the emergence and progression of the disease. The article describes how this information collectively, provides a framework for designing specific novel therapeutic approaches targeting the cell and tissue interactions driving tumour metastasis and its manifold effects on the whole body.

RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia.

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

An integrin alpha(v)beta(3)-c-Src oncogenic unit promotes anchorage-independence and tumor progression.

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.

Expression of melanocyte-related genes in human breast cancer and its implications.

We report the expression of melanocyte-related genes (MRG) in freshly resected, histopathologically confirmed, human breast cancer specimens and describe experiments illuminating similar observations on a variety of breast cancer cell lines including MDA-MB-435. This finding has implications for research on breast cancer, for clinical investigation of cancer patients presenting with metastases from occult primary tumors and for understanding aberrant differentiation in cancer cells. For example, higher expression of six MRG correlated inversely with propensity for metastatic spread in clones isolated from the human breast cancer cell line MDA-MB-435. Comparisons of MRG expression in cells growing in vitro with those seen in tumors generated by the same lines in vivo showed that the levels of activity of these genes are influenced by the surrounding environment. Also, silencing of expression of the melanocyte-related transcription factor MITF, by transduction of the non-metastatic clone NM2C5 with a construct expressing a specific anti-MITF shRNA, resulted in decreased production of 5 of the melanocyte-related proteins including TYRP1, Pmel 17, MART 1(Melan-A) and TYRP2, but no increase in metastatic capability. Hence MRG expression reproducibly ear-marked, but did not cause, metastatic incompetence. We also report cytogenetic and other data that conflict with the recent suggestion that MDA-MB-435 is of melanocytic origin and are more consistent with its original designation as being of mammary lineage. We conclude that detection of MRG expression profiles in freshly excised breast cancers and in cultured breast cancer cells reflects the operationally important clinical phenomenon of inappropriate gene expression in malignant neoplasms. Concomitantly, we suggest that the evidence we have obtained (i) collectively supports the continued widespread use of the MDA-MB-435 cell line in breast cancer and metastasis research and (ii) advances knowledge of the diversity of aberrant differentiation programs in malignant cells, which is valuable for making accurate diagnoses and treatment decisions in clinical oncology.

Metastasis-related plasma membrane proteins of human breast cancer cells identified by comparative quantitative mass spectrometry.

The spread of cancer cells from a primary tumor to form metastasis at distant sites is a complex multistep process. The cancer cell proteins and plasma membrane proteins in particular involved in this process are poorly defined, and a study of the very early events of the metastatic process using clinical samples or in vitro assays is not feasible. We have used a unique model system consisting of two isogenic human breast cancer cell lines that are equally tumorigenic in mice; but although one gives rise to metastasis, the other disseminates single cells that remain dormant at distant organs. Membrane purification and comparative quantitative LC-MS/MS proteomics identified 13 membrane proteins that were expressed at higher levels and three that were underexpressed in the metastatic compared with the non-metastatic cell line from a total of 1919 identified protein entries. Among the proteins were ecto-5'-nucleotidase (CD73), NDRG1, integrin beta1, CD44, CD74, and major histocompatibility complex class II proteins. The altered expression levels of proteins identified by LC-MS/MS were validated using flow cytometry, Western blotting, and immunocyto- and immunohistochemistry. Analysis of clinical breast cancer biopsies demonstrated a significant correlation between high ecto-5'-nucleotidase and integrin beta1 expression and poor outcome, measured as tumor spread or distant recurrence within a 10-year follow-up. Further the tissue analysis suggested that NDRG1, HLA-DRalpha, HLA-DRbeta, and CD74 were associated with the ER(-)/PR(-) phenotype represented by the two cell lines. The study demonstrates a quantitative and comparative proteomics strategy to identify clinically relevant key molecules in the early events of metastasis, some of which may prove to be potential targets for cancer therapy.

Osteopontin gene expression determines spontaneous metastatic performance of orthotopic human breast cancer xenografts.

A major problem in the therapeutic management of cancer is the growth of metastases in distant organs, but the genes orchestrating the process need to be identified for the rational design of new treatment. Here, we provide decisive experimental evidence demonstrating the causal involvement of a specific gene, osteopontin (OPN), in the pathogenesis of metastasis by human breast cancer cells and implicating some of its probable partners. Stable long-term depletion, or up-regulation, of OPN gene expression in a matched, isogenic pair of human breast cancer cell lines of differing metastatic proficiency reproducibly changed their ability to colonize distant organs. OPN down-regulation was achieved by transduction of the metastatic line with a DNA construct encoding a small hairpin RNA in a vector labeled with red fluorescent protein and resulted in a marked reduction of metastatic load (P < 0.01). Up-regulation of OPN in the negligibly metastatic line, with a green fluorescent protein-marked retroviral vector containing OPN cDNA driven by a strong promoter, resulted in heavy colonization of the lungs and lymph nodes (P < 0.005). The reciprocal changes in behavior of these matched cell lines cross-corroborate each other. Concomitant changes were seen in the expression of other metastasis-related genes in both modulated lines. The data indicate that therapeutic targeting of tumor OPN molecules could reset metastatically relevant gene networks, resulting in clinical benefit.

Gene expression profiling of human lymph node metastases and matched primary breast carcinomas: clinical implications.

The genetic program that drives tumor metastasis and the mode and timing of its initiation are of great practical significance to clinical management. Modern technical advances open new opportunities for gaining useful relevant information. Gene expression profiles of histologically-verified viable tissue from lymph node metastases were compared with those of matched primary breast cancers from 10 different patients, among samples from over 400 cases, using high-throughput oligonucleotide arrays comprising probes for 22,000 genes. It was observed that metastases have very similar expression signatures to their parent tumors. However, detailed computational analysis revealed that a small number of genes were consistently differentially expressed between 100% of tumors and metastases, suggesting that these are mechanistically important. Lists of such candidate genes, of potential clinical interest, are provided. We interpret these results in the framework of a meta-analysis of previous investigations by others and ourselves and of existing clinical knowledge on the behavior of human tumors. The collective data show that metastases resemble their primary tumors but the signatures obtained in different studies are not sufficiently reproducible or informative to be prognostically useful, although they do give valuable insights into the pathogenesis and biology of human tumor metastasis. The findings indicate that the genetic program encoding metastasis is implemented progressively over time although, occasionally, this evolution can occur rapidly, early in the life of the neoplasm. The important clinical significance of this deduction is that, in most patients, early detection provides time for appropriate therapeutic intervention to be effective in obstructing metastasis.

Dormant cancer cells retrieved from metastasis-free organs regain tumorigenic and metastatic potency.

This study shows that solitary, dormant human cancer cells, retrieved from metastasis-free organs of animals carrying spontaneously metastatic primary tumors, can reactivate their tumorigenic and metastatic potency. The tumors were produced by MDA-MB-435 CL16 breast cancer cells permanently labeled with green fluorescent protein and the neomycin resistance gene. This enabled unequivocal identification of tumor cells emerging from organ explants cultured in neomycin to eliminate nonneoplastic host cells. Rescued cells resumed proliferation and generated lines that were tumorigenic and metastatic in fresh animals. All resulting primary and secondary tumors were uniformly labeled. Cells recovered from bone marrows and spleens, where there were no metastases, were as tumorigenic and metastatic as cells recovered from lungs and lymph nodes, which are the preferred sites of colonization for this tumor line. This evidence that malignant growth of disseminated cancer cells is suspended indefinitely by microenvironmental conditions in metastasis-free organs, although it is still active in others of the same host, shows that neoplastic progression can be arrested and has far-reaching biological and clinical implications. Specifically, it predicts the existence of natural, nonimmune host mechanisms that stimulate or inactivate tumor growth in different anatomical sites, which may be exploitable for therapeutic benefit.

Effect of the reduction of superoxide dismutase 1 and 2 or treatment with alpha-tocopherol on tumorigenesis in Atm-deficient mice.

Atm-deficient mice, a cancer-prone model of the human disease ataxia-telangiectasia, display increased levels of oxidative stress and damage. Chronic treatment of these mice with the nitroxide antioxidant and superoxide dismutase (SOD) mimetic Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) resulted in an increased latency to tumorigenesis. We initially hypothesized that the chemopreventative effect of Tempol was due to its SOD mimetic activity reducing cellular oxidative stress and damage. However, it is also possible that the chemopreventative effect of Tempol results from mechanisms other than directly reducing superoxide radical-induced oxidative stress and damage. To help distinguish between these possibilities, we attempted to genetically increase oxidative stress in Atm-deficient mice by either removing cytosolic Sod1 or reducing mitochondrial Sod2, or we attempted to decrease oxidative stress by treatment of Atm-deficient mice with alpha-tocopherol. Surprisingly, we found that reducing both Atm and Sod1 or Atm and Sod2 did not shorten latency to tumorigenesis or significantly affect life span. Furthermore, continuous administration of alpha-tocopherol did not affect latency to thymic lymphomas. Thus, genetically reducing Sod in Atm-deficient mice or treatment with alpha-tocopherol had no effect on survival or tumorigenesis, suggesting that the chemopreventative effect of Tempol may be at least partially independent of its effects on reducing oxidative damage and stress.

Tumor-stromal interactions reciprocally modulate gene expression patterns during carcinogenesis and metastasis.

This study used a unique xenogeneic breast cancer model to study the effects of tumor cells and neighboring host cells upon each other in tumor growth and metastasis. It exploited species differences between the interacting components to determine how the host influenced the tumor and vice versa. It was found that the gene expression profiles of highly and poorly metastatic clones from the same human breast carcinoma changed differentially when the cells were transferred from growth in vitro to the mammary gland. We describe novel sets of genes, validated by human-specific probes, which were induced in the 2 isogenic, but phenotypically different, tumor lineages by the mammary environment. Conversely, the tumor cells also induced changes in gene expression in the neighboring host stromal (i.e., mesenchymal) cell lineages, validated by mouse-specific probes. Reciprocal inductive interactions were also demonstrated in the tumor deposits formed preferentially in the lungs and lymph nodes by the highly metastatic tumor cells. Subtraction of the induced gene changes in the primary site from those in the metastases revealed that the number and magnitude of specific gene inductions in colonized organs were moderate. This finding indicates that the gene expression program causing metastasis has only limited flexibility and fits well with clinical observations that tumor cells form metastases preferentially in select organs, although tumor cells are scattered ubiquitously. This dependency on suitable host niches suggests new molecular therapeutic avenues that target genes in the host-support system that is manipulated by the malignant cells.