Lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.

The International Agency for Research on Cancer determined that obesity is the primary preventable cause of breast cancer. The nuclear receptor peroxisome proliferator activated receptor γ (PPARγ) binds inflammatory mediators in obesity and its expression is reduced in human breast cancer. We created a new model to better understand how the obese microenvironment alters nuclear receptor function in breast cancer. The obesity related cancer phenotype was PPARγ dependent; deletion of PPARγ in mammary epithelium which is a tumor suppressor in lean mice unexpectedly increased tumor latency, reduced the luminal progenitor (LP) tumor cell fraction, and increased autophagic and senescent cells. Loss of PPARγ expression in mammary epithelium of obese mice increased expression of 2-aminoadipate semialdehyde synthase (AASS) which regulates lysine catabolism to acetoacetate. PPARγ-associated co-repressors and activators regulated AASS expression via a canonical response element. AASS expression was significantly reduced in human breast cancer, and AASS overexpression or acetoacetate treatment inhibited proliferation and induced autophagy and senescence in human breast cancer cell lines. Genetic or pharmacologic HDAC inhibition promoted autophagy and senescence in mammary tumor cells in vitro and in vivo. We concluded that lysine metabolism is a novel metabolic tumor suppressor pathway in breast cancer.

PARP5B is required for nonhomologous end joining during tumorigenesis in vivo.

Poly(ADP-ribose) polymerases (PARP) act as DNA damage sensors that produce poly(ADP-ribose) (PAR) chains at double-strand breaks, facilitating the recruitment of repair factors. Cancers with homologous recombination defects are sensitive to small molecule PARP inhibitors. Despite PARP5B gene copy number changes in many cancers, the effects of this genetic alteration on tumor phenotype are largely unknown. To better understand this clinical finding, we characterized a PARP5B null mutation in a carcinogen-induced in vivo head and neck squamous cell carcinoma (SCC) model. Reduced PARP5B expression inhibited tumor growth, induced primary tumor differentiation and apoptosis, and inhibited cell proliferation and metastasis. Loss of PARP5B expression-induced ataxia telangiectasia and Rad3 related (ATR) activation and depleted the cancer stem cell fraction. PARP5B null tumor cells lacked 53BP1+ double-strand break foci, ATM activation, and p53 induction compared to PARP5B+/+ cancers. PARP5B null SCC expresses a multiprotein complex containing PML, pRPA, Rad50, Rad51, XRCC1, proliferating cell nuclear antigen (PCNA), and Mcm2, suggesting an HR-mediated repair mechanism at DNA replication foci. Low doses of etoposide combined with the PARP5B inhibitor XAV939 induced senescence and apoptosis in human SCC lines. NBS1 overexpression in these cells inhibited the effects of low-dose etoposide/XAV939 treatment. Our results indicate that PARP5B inhibition is new targeted cancer therapy.

DNA Double-strand Break Signaling Is a Therapeutic Target in Head and Neck Cancer.

BACKGROUND: Head and neck cancer (HNC) is common worldwide. Given poor outcomes for patients with HNC, research into targeted therapies is needed. Ataxia telangiectasia mutated (ATM) is a DNA damage kinase which is activated by double-strand DNA breaks. We tested the effects of a novel ATM inhibitor on HNC cell lines and xenografts. MATERIALS AND METHODS: p53-Binding protein 1 and phosphorylated ATM were localized in cultured cells by immunofluorescence microscopy. Protein expression was determined by western blot. Tumor xenografts were established by injecting HNC lines into immunocompromised mice. Tumor sections were characterized by immunohistochemistry. Apoptotic cells were determined by terminal transferase-mediated dUTP nick-end labeling assay. RESULTS: ATM inhibition increased double-strand DNA breaks at replication foci in HNC cell lines. ATM inhibition affected cell-cycle regulatory protein expression, blocked cell-cycle progression at the G2/M phase and resulted in apoptosis. CONCLUSION: ATM inhibition may be therapeutically useful in treating HNC.

Telomere DNA Damage Signaling Regulates Prostate Cancer Tumorigenesis.

Telomere shortening has been demonstrated in benign prostatic hypertrophy (BPH), which is associated with prostate epithelial cell senescence. Telomere shortening is the most frequently observed genetic alteration in prostatic intraepithelial neoplasia, and is associated with poor clinical outcomes in prostate cancer. Gene expression database analysis revealed decreased TRF2 expression during malignant progression of the prostate gland. We reasoned that reduced TRF2 expression in prostate epithelium, by activating the telomere DNA damage response, would allow us to model both benign and malignant prostate disease. Prostate glands with reduced epithelial TRF2 expression developed age- and p53-dependent hypertrophy, senescence, ductal dilation, and smooth muscle hyperplasia similar to human BPH. Prostate tumors with reduced TRF2 expression were classified as high-grade androgen receptor-negative adenocarcinomas, which exhibited decreased latency, increased proliferation, and distant metastases. Prostate cancer stem cells with reduced TRF2 expression were highly tumorigenic and maintained telomeres both by telomerase and alternative lengthening (ALT). Telomerase inhibition in prostate glands with reduced TRF2 expression produced significant reduction in prostate tumor incidence by halting progression at intraepithelial neoplasia (PIN). These lesions were highly differentiated, exhibited low proliferation index, and high apoptotic cell fraction. Prostate tumors with reduced TRF2 expression and telomerase inhibition failed to metastasize and did not exhibit ALT. IMPLICATIONS: Our results demonstrate that the telomere DNA damage response regulates BPH, PIN, and prostate cancer and may be therapeutically manipulated to prevent prostate cancer progression.

Paracrine Interaction of Cancer Stem Cell Populations Is Regulated by the Senescence-Associated Secretory Phenotype (SASP).

Dyskeratosis congenita is a telomere DNA damage syndrome characterized by defective telomere maintenance, bone marrow failure, and increased head and neck cancer risk. The Pot1b-/-;Terc+/- mouse exhibits some features of dyskeratosis congenita, but head and neck cancer was not reported in this model. To model the head and neck cancer phenotype, we created unique Pot1b- and p53-null-mutant models which allow genetic lineage tracing of two distinct stem cell populations. Loss of Pot1b expression depleted stem cells via ATR/Chk1/p53 signaling. Tumorigenesis was inhibited in Pot1b-/-;p53+/+ mice due to cellular senescence. Pot1b-/-;p53-/- tumors also exhibited senescence, but proliferated and metastasized with expansion of Lgr6+ stem cells indicative of senescence-associated secretory phenotype. Selective depletion of the small K15+ stem cell fraction resulted in reduction of Lgr6+ cells and inhibition of tumorigenesis via senescence. Gene expression studies revealed that K15+ cancer stem cells regulate Lgr6+ cancer stem cell expansion via chemokine signaling. Genetic ablation of the chemokine receptor Cxcr2 inhibited cancer stem cell expansion and tumorigenesis via senescence. The effects of chemokines were primarily mediated by PI3K signaling, which is a therapeutic target in head and neck cancer. IMPLICATIONS: Paracrine interactions of cancer stem cell populations impact therapeutic options and patient outcomes.

Molecular and cellular basis of mammary gland fibrosis and cancer risk.

Mammary gland luminal cells are maintained by the proliferation of ER- luminal progenitor (LP) cells. Human breast LP cells exhibit telomere DNA damage, which is associated with mammographic density and increased cancer risk. Telomeric repeat factor 2 (TRF2) protects telomeres from DNA damage response. TRF2 expression is reduced in human breast cancers. We deleted TRF2 expression in mammary gland epithelium. Mammary glands lacking TRF2 expression exhibited increased telomere DNA damage response, histopathological and functional degeneration, and prominent ductal fibrosis. TRF2-deficient mammary tumors exhibited rapid onset and increased proliferation. Tumor derived LP cells failed to form tumors after transplantation. The MSC population was highly tumorigenic and maintained telomeres via the ALT mechanism. Telomere DNA damage response in mammary tumors resulted in p53 dependent ER+ cellular differentiation and sensitivity to anti-estrogen therapy. Our results provide a new in vivo model of mammographic density, stem cell differentiation, cancer risk, and therapeutic sensitivity.

Telomere DNA damage signaling regulates cancer stem cell evolution, epithelial mesenchymal transition, and metastasis.

Chromosome ends are protected by telomeres that prevent DNA damage response and degradation. When telomeres become critically short, the DNA damage response is activated at chromosome ends which induces cellular senescence or apoptosis. Telomeres are protected by the double stranded DNA binding protein TRF2 and maintained by telomerase or a recombination based mechanism known as alternative lengthening of telomeres (ALT). Telomerase is expressed in the basal layer of the epidermis, and stem cells in epidermis have longer telomeres than proliferating populations. Stem cell expansion has been associated with epithelial-mesenchymal transition (EMT) in cancer. EMT is a critical process in cancer progression in which cells acquire spindle morphology, migrate from the primary tumor, and spread to distant anatomic sites. Our previous study demonstrated that loss of TRF2 expression observed in human squamous cell carcinomas expanded metastatic cancer stem cells during mouse skin carcinogenesis. To determine if telomerase inhibition could block the TRF2-null mediated expansion of metastatic clones, we characterized skin carcinogenesis in a conditional TRF2/Terc double null mutant mouse. Loss of TRF2 and Terc expression resulted in telomere DNA damage, severely depleted CD34 + and Lgr6+ cancer stem cells, and induced terminal differentiation of metastatic cancer cells. However a novel cancer stem cell population evolved in primary tumors exhibiting genomic instability, ALT, and EMT. Surprisingly we discovered that metastatic clones evolved prior to histopathologic onset of primary tumors. These results have important implications for understanding the evolution and treatment of metastatic cancer.

The histone methyltransferase EZH2 promotes mammary stem and luminal progenitor cell expansion, metastasis and inhibits estrogen receptor-positive cellular differentiation in a model of basal breast cancer.

Mammary stem cells (MSCs) are the progenitor population for human breast epithelia. MSCs give rise during mammary gland development to estrogen receptor (ER)-negative basal cells and the ER- luminal progenitor (LP) population which maintains ER+ and ER- luminal cells. The MSC population is expanded and tumorigenic in some mouse mammary cancer models, and these tumor-initiating cells have been isolated from human breast cancers. MSC expansion is associated with aggressive biological behavior in human breast cancer. The LP population is tumorigenic in some mouse mammary cancer models, and is the progenitor population of basal breast cancer in humans. The enhancer of zeste homolog 2 (EZH2) is a methyltransferase which catalyzes lysine 27 methylation in histone H3 resulting in suppression of target gene expression. The histone demethylase JMJD3 opposes the activity of EZH2 by demethylating histone H3 lysine 27. EZH2 is a member of the polycomb group of proteins which regulates cell type identity. EZH2 expression was found to be increased in histologically normal human breast tissue among women with high breast cancer risk, and was elevated in ductal hyperplasia and ductal carcinoma in situ. EZH2 overexpression is associated with poorly differentiated and aggressive breast cancer in humans. However, the mechanisms by which EZH2 results in increased breast cancer risk and aggressive tumors are not completely characterized. Using in vivo transplantation of mammary cancer stem cells transduced with EZH2 or JMJD3 shRNAs, we demonstrated that EZH2 promotes mammary stem and LP cell expansion, metastasis and inhibits ER-positive cellular differentiation.

Role of microRNA-138 as a potential tumor suppressor in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive life-threatening disease associated with high mortality rates. While efforts have been made to explore the molecular mechanisms that contribute to the initiation and progression of HNSCC, most studies focus on protein-coding genes. Understanding of the genomic aberrations associated with noncoding genes (such as microRNAs) and their effects on HNSCC is still relatively limited. Recent evidence suggests that deregulation of microRNA genes (such as downregulation of miR-138) plays an important role in HNSCC. While deregulation of miR-138 has been frequently observed in HNSCC and other cancer types, the precise roles of miR-138 in tumorigenesis remain elusive. Recent bioinformatics analyses and functional studies using in vitro and in vivo systems have identified a number of functional targets for miR-138. These include genes that participate in essential biological processes that are highly relevant to the initiation and progression of HNSCC, including cell migration, epithelial to mesenchymal transition, cell cycle progression, DNA damage response and repair, senescence, and differentiation. However, the biological systems, study design, and data interpretation from these studies are highly variable, which hinder our understanding of the role of miR-138 in tumorigenesis at molecular level. In this review, we will first introduce the significance of microRNA deregulation in HNSCC. We will then provide a comprehensive review and integrative analysis of the existing studies on miR-138, and aim to define its molecular mechanisms that contribute to the initiation and progression of HNSCC.

Regulation of the Nijmegen breakage syndrome 1 gene NBS1 by c-myc, p53 and coactivators mediates estrogen protection from DNA damage in breast cancer cells.

In mammalian cells more than 90% of double-strand breaks are repaired by NHEJ. Impairment of this pathway is associated with cell cycle arrest, cell death, genomic instability and cancer. Human diseases such as Nijmegen breakage syndrome, due to mutations in the NBS1 gene, produce defects in resection of double-strand breaks. NBS1 hypomorphic mutant mice are viable, and cells from these mice are defective in S phase and G2/M checkpoints. NBS1 polymorphisms have been associated with increased risk of breast cancer. We previously demonstrated that estradiol protected estrogen receptor (ER)-positive (+) breast cancer cell lines against double-strand breaks and cell death. We now demonstrate that protection from double-strand break damage in ER+ cells is mediated via regulation by c-myc, p53, CBP and SRC1 coactivators in intron 1 of the NBS1 gene. We concluded that NBS1 is responsible for estradiol-mediated protection from double-strand breaks in ER+ breast cancer cells.

Dysfunctional telomeres promote genomic instability and metastasis in the absence of telomerase activity in oncogene induced mammary cancer.

Telomerase is a ribonucleoprotein that maintains the ends of chromosomes (telomeres). In normal cells lacking telomerase activity, telomeres shorten with each cell division because of the inability to completely synthesize the lagging strand. Critically shortened telomeres elicit DNA damage responses and limit cellular division and lifespan, providing an important tumor suppressor function. Most human cancer cells express telomerase which contributes significantly to the tumor phenotype. In human breast cancer, telomerase expression is predictive of clinical outcomes such as lymph node metastasis and survival. In mouse models of mammary cancer, telomerase expression is also upregulated. Telomerase overexpression resulted in spontaneous mammary tumor development in aged female mice. Increased mammary cancer also was observed when telomerase deficient mice were crossed with p53 null mutant animals. However, the effects of telomerase and telomere length on oncogene driven mammary cancer have not been completely characterized. To address these issues we characterized neu proto-oncogene driven mammary tumor formation in G1 Terc-/- (telomerase deficient with long telomeres), G3 Terc-/- (telomerase deficient with short telomeres), and Terc+/+ mice. Telomerase deficiency reduced the number of mammary tumors and increased tumor latency regardless of telomere length. Decreased tumor formation correlated with increased apoptosis in Terc deficient tumors. Short telomeres dramatically increased lung metastasis which correlated with increased genomic instability, and specific alterations in DNA copy number and gene expression. We concluded that short telomeres promote metastasis in the absence of telomerase activity in neu oncogene driven mammary tumors.

Haploinsufficiency of the Nijmegen breakage syndrome 1 gene increases mammary tumor latency and metastasis.

Human diseases such as Nijmegen breakage syndrome due to mutations in the NBS1 gene result in defects in resection of double strand breaks. NBS1 functions as part of the MRN complex which functions in homologous recombination and non-homologous end joining. NBS is a rare human autosomal recessive disorder caused by hypomorphic mutations. At the cellular level, NBS is characterized by radiosensitivity, chromosomal breakage and defective cell cycle checkpoints. NBS1 null mutations result in early embryonic lethality in mice, but NBS1 hypomorphic mutants are viable. Cells from these mice are defective in S phase and G2/M checkpoints. In humans, NBS1 polymorphisms have been associated with increased risk of breast cancer. MRN expression was reduced in the majority of breast tumors, and low expression of MRN correlated with increased histologic grade and estrogen receptor negativity. While these studies have shown NBS1 to be important in clinical outcomes of patients with breast cancer, mammary tumors are rare in the NBS1 haploinsufficient mouse. To better understand the role of NBS1 in mammary tumorigenesis, we examined the NBS1+/-;MMTV-neu mouse model. Mammary tumor latency was markedly increased in NBS1+/-;neu mice compared to NBS1+/+;neu control animals. This effect was due to increased apoptosis in early NBS1+/-;neu mammary tumors. However, NBS1+/-;neu mammary tumors were highly metastatic and demonstrated clear differences in gene expression profiles compared to control tumors. We concluded that NBS1 haploinsufficiency results in increased mammary tumor latency and metastasis.

Telomere dysfunction promotes metastasis in a TERC null mouse model of head and neck cancer.

Squamous cell carcinoma arises from highly proliferative basal layer epithelial cells, which normally divide for a short time before detaching from the basement membrane and undergoing terminal differentiation. Basal layer cells in stratified epithelia express the reverse transcriptase known as telomerase. Most human cells do not express telomerase and therefore are subject to loss of telomeric DNA with age due to the inability of lagging strand synthesis to completely replicate chromosomal ends. Late generation telomerase deficient mice exhibit signs of premature aging including reduced function of proliferating cellular compartments. We examined development of squamous cell carcinoma in a telomerase deficient murine background with long and short telomeres. G1 Terc-/- mice (long telomeres) had fewer lymph node metastases, which correlated with increased numbers of apoptotic cells in these tumors compared with wild-type mice. However, G5 Terc-/- mice with short telomeres had increased metastatic tumor burden similar to wild type mice. This increased metastasis correlated with genomic instability and aneuploidy in tumor cells from G5 Terc-/- mice. A number of similarities with human SCC were noted in the mouse model, and dramatic differences in global gene expression profiles were shown between primary and metastatic tumors. We concluded that telomere shortening promotes metastatic tumor development in a Terc null mouse model of head and neck cancer.

Resistance to telomerase inhibition by human squamous cell carcinoma cell lines.

Telomeres are nucleoprotein structures at the ends of chromosomes that are composed of a repetitive G rich sequence and telomeric binding proteins. Telomeres prevent the degradation of chromosomal ends and protect against inappropriate recombination. Telomere attrition involves a tumor suppressor pathway that limits the replication of premalignant cells. The loss of telomeric DNA with each round of replication leads to growth arrest accompanied by senescence or apoptosis. Many tumor cells activate the telomerase gene to bypass senescence. Telomerase is a multisubunit ribonucleoprotein that uses an RNA template to catalyze the addition of telomeric DNA to chromosomal ends. Overexpression of the TERT subunit leads to telomere lengthening and extension of the replicative lifespan. Dominant-negative telomerase has been shown to inhibit telomerase activity in many tumor cell lines, and this is associated with telomere shortening and apoptosis. Additionally, pharmacological telomerase inhibitors have been developed which lead to progressive telomere shortening and programmed cell death. In this study, we report a series of human squamous cell carcinoma cell lines that have high telomerase activity and short telomeres. Dominant-negative telomerase expression and pharmacological telomerase inhibition failed to completely inhibit enzymatic activity which was accompanied by the lack of telomere shortening. These cells continued to proliferate and demonstrated fewer responsive genes when treated with a pharmacological telomerase inhibitor. We concluded that some human squamous cell carcinoma cell lines are resistant to telomerase inhibition.

Steroid receptor coactivator 1 deficiency increases MMTV-neu mediated tumor latency and differentiation specific gene expression, decreases metastasis, and inhibits response to PPAR ligands.

BACKGROUND: The peroxisome proliferator activated receptor (PPAR) subgroup of the nuclear hormone receptor superfamily is activated by a variety of natural and synthetic ligands. PPARs can heterodimerize with retinoid X receptors, which have homology to other members of the nuclear receptor superfamily. Ligand binding to PPAR/RXRs results in recruitment of transcriptional coactivator proteins such as steroid receptor coactivator 1 (SRC-1) and CREB binding protein (CBP). Both SRC-1 and CBP are histone acetyltransferases, which by modifying nucleosomal histones, produce more open chromatin structure and increase transcriptional activity. Nuclear hormone receptors can recruit limiting amounts of coactivators from other transcription factor binding sites such as AP-1, thereby inhibiting the activity of AP-1 target genes. PPAR and RXR ligands have been used in experimental breast cancer therapy. The role of coactivator expression in mammary tumorigenesis and response to drug therapy has been the subject of recent studies. METHODS: We examined the effects of loss of SRC-1 on MMTV-neu mediated mammary tumorigenesis. RESULTS: SRC-1 null mutation in mammary tumor prone mice increased the tumor latency period, reduced tumor proliferation index and metastasis, inhibited response to PPAR and RXR ligands, and induced genes involved in mammary gland differentiation. We also examined human breast cancer cell lines overexpressing SRC-1 or CBP. Coactivator overexpression increased cellular proliferation with resistance to PPAR and RXR ligands and remodeled chromatin of the proximal epidermal growth factor receptor promoter. CONCLUSIONS: These results indicate that histone acetyltransferases play key roles in mammary tumorigenesis and response to anti-proliferative therapies.

Jun amino-terminal kinase 1 activation promotes cell survival in ErbB2-positive breast cancer.

BACKGROUND: Downstream signaling is a key component of Her2/neu overexpression in human breast cancer. Major survival pathways downstream of Her2/neu include mitogen and stress activated protein kinases (ERK, JNK, p38). MATERIALS AND METHODS: MAPK protein expression was examined in mouse and human cancer tissue. MAPK expression was inhibited by genetic and pharmacologic methods in human breast cancer cell lines. The effects of MAPK inhibition on tumor formation in a preclinical model were determined. RESULTS: It was shown that tumors from MMTV-neu mice expressed high levels of activated JNK1. Levels of this kinase were also highest in Her2/neu overexpressing human breast cancer cell lines. JNK1 inhibition specifically induced apoptosis in these lines. A JNK1 inhibitor also increased the latency period and decreased growth of MMTV-neu tumors by induction of apoptosis. JNK1 was preferentially activated in human breast cancer tissue overexpressing Her2/neu. CONCLUSION: JNK1 promotes cell survival in Her2/neu-positive breast cancer.

Deregulation of manganese superoxide dismutase (SOD2) expression and lymph node metastasis in tongue squamous cell carcinoma.

BACKGROUND: Lymph node metastasis is a critical event in the progression of tongue squamous cell carcinoma (TSCC). The identification of biomarkers associated with the metastatic process would provide critical prognostic information to facilitate clinical decision making. Previous studies showed that deregulation of manganese superoxide dismutase (SOD2) expression is a frequent event in TSCC and may be associated with enhanced cell invasion. The purpose of this study is to further evaluate whether the expression level of SOD2 is correlated with the metastatic status in TSCC patients. METHODS: We first examined the SOD2 expression at mRNA level on 53 TSCC and 22 normal control samples based on pooled-analysis of existing microarray datasets. To confirm our observations, we examined the expression of SOD2 at protein level on an additional TSCC patient cohort (n = 100), as well as 31 premalignant dysplasias, 15 normal tongue mucosa, and 32 lymph node metastatic diseases by immunohistochemistry (IHC). RESULTS: The SOD2 mRNA level in primary TSCC tissue is reversely correlated with lymph node metastasis in the first TSCC patient cohort. The SOD2 protein level in primary TSCC tissue is also reversely correlated with lymph node metastasis in the second TSCC patient cohort. Deregulation of SOD2 expression is a common event in TSCC and appears to be associated with disease progression. Statistical analysis revealed that the reduced SOD2 expression in primary tumor tissue is associated with lymph node metastasis in both TSCC patient cohorts examined. CONCLUSIONS: Our study suggested that the deregulation of SOD2 in TSCC has potential predictive values for lymph node metastasis, and may serve as a therapeutic target for patients at risk of metastasis.

Embryoid body attachment to reconstituted basement membrane induces a genetic program of epithelial differentiation via jun N-terminal kinase signaling.

Embryonic stem (ES) cells are derived from early stage mammalian embryos and have broad developmental potential. These cells can be manipulated experimentally to generate cells of multiple tissue types which could be important in treating human diseases. The ability to produce relevant amounts of these differentiated cell populations creates the basis for clinical interventions in tissue regeneration and repair. Understanding how embryonic stem cells differentiate also can reveal important insights into cell biology. A previously reported mouse embryonic stem cell model demonstrated that differentiated epithelial cells migrated out of embryoid bodies attached to reconstituted basement membrane. We used genomic technology to profile ES cell populations in order to understand the molecular mechanisms leading to epithelial differentiation. Cells with characteristics of cultured epithelium migrated from embryoid bodies attached to reconstituted basement membrane. However, cells that comprised embryoid bodies also rapidly lost ES cell-specific gene expression and expressed proteins characteristic of stratified epithelia within hours of attachment to basement membrane. Gene expression profiling of sorted cell populations revealed upregulation of the BMP/TGFbeta signaling pathway, which was not sufficient for epithelial differentiation in the absence of basement membrane attachment. Activation of c-jun N-terminal kinase 1 (JNK1) and increased expression of Jun family transcription factors was observed during epithelial differentiation of ES cells. Inhibition of JNK signaling completely blocked epithelial differentiation in this model, revealing a key mechanism by which ES cells adopt epithelial characteristics via basement membrane attachment.

Chronologic aging decreases tumor angiogenesis and metastasis in a mouse model of head and neck cancer.

The incidence of malignant tumors increases with age. This may be due to the duration of carcinogenesis or age related changes providing a favorable environment for tumor formation. Aging is associated with molecular, cellular and physiological events that influence carcinogenesis and cancer growth. Physiologic cell proliferation, differentiation, and aging can result in cell death. However, under the influence of exogenous or endogenous factors cells can undergo pathologic dedifferentiation, immortalization, and neoplastic clone formation. The effects of age have been recognized in both animal and human malignancies. These processes can result in cellular senescence as a barrier to tumorigenesis. Inducing senescence is an important outcome for the successful treatment of cancers particularly those resistant to apoptosis. Senescence is associated with polyploidy in several human cell lines. Polyploid cells are dangerous in that they can undergo aberrant mitoses giving rise to unstable progeny. Polyploid cells have been shown to escape senescence and divide. We examined the effects of aging on squamous cell carcinoma formation in a mouse model. Chronologically aged mice experience shorter tumor latency periods than wild-type animals. Tumors in aged mice were poorly vascularized, necrotic, and produced significantly fewer cervical lymph node metastases. Vascular endothelial growth factor expression was similar in primary tumors from young and old mice, but microvessel density was significantly reduced in tumors arising in aged mice. These results indicate that host response to angiogenic factors inhibit tumor growth and metastasis of head and neck cancer.

Impaired T lymphocyte function increases tumorigenicity and decreases tumor latency in a mouse model of head and neck cancer.

Squamous cell carcinoma of the head and neck (HNSCC) is the sixth most frequent cancer worldwide. SCC is the most common malignant tumor of the oral cavity with over 35,000 cases and 8,000 deaths reported in the United States each year. Previous case studies have reported increased incidence of HNSCC in patients on immunosuppressive therapy for organ transplantation. The results of these studies indicate that effective immune surveillance is important for preventing emergence of HNSCC. HNSCC may also inhibit immune response to tumor cells, which may be responsible for progression. We previously reported induction of metastatic HNSCC in p53 null mutant mice. Despite induction with the potent carcinogen dimethylbenzanthracene, each mouse developed only 1-2 primary tumors with a relatively long induction period of 22 weeks. We hypothesized that immune surveillance might eliminate early tumor cells resulting in the relatively small number of primary tumors and long induction time. To test this hypothesis we performed the induction protocol in nude mice which have defective T lymphocyte function. Decreased T lymphocyte function resulted in reduced tumor latency and increased tumor formation. Immunohistochemical studies showed that expression of cell cycle regulatory proteins is similar in mouse and human HNSCC. However, distinct differences exist between primary and metastatic tumors from nude and wild-type mice. We also determined that lymphocytes react to metastatic tumor cells by upregulating immunoglobin gene expression but are prone to apoptosis via decreased expression of survival factors and upregulation of cell death genes.