Peter J. Hornsby

Senescent Human Fibroblasts Increase the Early Growth of Xenograft Tumors via Matrix Metalloproteinase Secretion

Although cellular senescence is believed to have a tumor suppressor function, senescent cells have been shown to increase the potential for growth of adjacent cancer cells in animal models. Replicatively senescent human fibroblasts increase the growth of cotransplanted cancer cells in vivo, but the role of cells that have undergone damage-mediated stress-induced premature senescence (SIPS) has not been studied in mouse transplant models. Here, we show that human fibroblasts that have undergone SIPS by exposure to the DNA-damaging agent bleomycin increase the growth of cotransplanted cancer cells (MDA-MB-231) in immunodeficient mice. Xenografts containing SIPS fibroblasts (SIPSF) exhibited early tissue damage as evidenced by fluid accumulation (edema). Cancer cells adjacent to the fluid showed increased DNA synthesis. Fluid accumulation, increased xenograft size, and increased cell proliferation were all reduced by the matrix metalloproteinase (MMP) inhibitor GM6001. MMPs and other genes characteristic of inflammation/tissue injury were overexpressed in SIPSF. Inhibition of MMP activity did not affect SIPSF stimulation of cancer cell proliferation in culture. However, another overexpressed product (hepatocyte growth factor) did have a direct mitogenic action on cancer cells. Based on the present results, we propose that senescent cells may promote cancer growth both by a direct mitogenic effect and by an indirect effect via tissue damage. Senescent stromal cells may cause an MMP-mediated increase in permeability of adjacent capillaries, thereby exposing incipient cancer cells to increased levels of mitogens, cytokines, and other plasma products. This exposure may increase cancer cell proliferation and result in promotion of preneoplastic cells.

Interleukin 6 secreted from adipose stromal cells promotes migration and invasion of breast cancer cells.

Excessive adiposity has long been associated with increased incidence of breast cancer in post-menopausal women, and with increased mortality from breast cancer, regardless of the menopausal status. Although adipose tissue-derived estrogen contributes to obesity-associated risk for estrogen receptor (ER)-positive breast cancer, the estrogen-independent impact of adipose tissue on tumor invasion and progression needs to be elucidated. Here, we show that adipose stromal cells (ASCs) significantly stimulate migration and invasion of ER-negative breast cancer cells in vitro and tumor invasion in a co-transplant xenograft mouse model. Our study also identifies cofilin-1, a known regulator of actin dynamics, as a determinant of the tumor-promoting activity of ASCs. The cofilin-1-dependent pathway affects the production of interleukin 6 (IL-6) in ASCs. Depletion of IL-6 from the ASC-conditioned medium abrogated the stimulatory effect of ASCs on the migration and invasion of breast tumor cells. Thus, our study uncovers a link between a cytoskeleton-based pathway in ASCs and the stromal impact on breast cancer cells.

Resistance to experimental tumorigenesis in cells of a long‐lived mammal, the naked mole‐rat (Heterocephalus glaber)

The naked mole‐rat (NMR, Heterocephalus glaber) is a long‐lived mammal in which spontaneous cancer has not been observed. To investigate possible mechanisms for cancer resistance in this species, we studied the properties of skin fibroblasts from the NMR following transduction with oncogenes that cause cells of other mammalian species to form malignant tumors. Naked mole‐rat fibroblasts were transduced with a retrovirus encoding SV40 large T antigen and oncogenic RasG12V. Following transplantation of transduced cells into immunodeficient mice, cells rapidly entered crisis, as evidenced by the presence of anaphase bridges, giant cells with enlarged nuclei, multinucleated cells, and cells with large number of chromosomes or abnormal chromatin material. In contrast, similarly transduced mouse and rat fibroblasts formed tumors that grew rapidly without crisis. Crisis was also observed after > 40 population doublings in SV40 TAg/Ras‐expressing NMR cells in culture. Crisis in culture was prevented by additional infection of the cells with a retrovirus encoding hTERT (telomerase reverse transcriptase). SV40 TAg/Ras/hTERT‐expressing NMR cells formed tumors that grew rapidly in immunodeficient mice without evidence of crisis. Crisis could also be induced in SV40 TAg/Ras‐expressing NMR cells by loss of anchorage, but after hTERT transduction, cells were able to proliferate normally following loss of anchorage. Thus, rapid crisis is a response of oncogene‐expressing NMR cells to growth in an in vivo environment, which requires anchorage independence, and hTERT permits cells to avoid crisis and to achieve malignant tumor growth. The unique reaction of NMR cells to oncogene expression may form part of the cancer resistance of this species.

Genomic Loss of Tumor Suppressor miRNA-204 Promotes Cancer Cell Migration and Invasion by Activating AKT/mTOR/Rac1 Signaling and Actin Reorganization

Increasing evidence suggests that chromosomal regions containing microRNAs are functionally important in cancers. Here, we show that genomic loci encoding miR-204 are frequently lost in multiple cancers, including ovarian cancers, pediatric renal tumors, and breast cancers. MiR-204 shows drastically reduced expression in several cancers and acts as a potent tumor suppressor, inhibiting tumor metastasis in vivo when systemically delivered. We demonstrated that miR-204 exerts its function by targeting genes involved in tumorigenesis including brain-derived neurotrophic factor (BDNF), a neurotrophin family member which is known to promote tumor angiogenesis and invasiveness. Analysis of primary tumors shows that increased expression of BDNF or its receptor tropomyosin-related kinase B (TrkB) parallel a markedly reduced expression of miR-204. Our results reveal that loss of miR-204 results in BDNF overexpression and subsequent activation of the small GTPase Rac1 and actin reorganization through the AKT/mTOR signaling pathway leading to cancer cell migration and invasion. These results suggest that microdeletion of genomic loci containing miR-204 is directly linked with the deregulation of key oncogenic pathways that provide crucial stimulus for tumor growth and metastasis. Our findings provide a strong rationale for manipulating miR-204 levels therapeutically to suppress tumor metastasis.

Generation of induced pluripotent stem cells from newborn marmoset skin fibroblasts.

Induced pluripotent stem cells (iPSCs) hold great promise for regenerative medicine. For the application of iPSCs to forms of autologous cell therapy, suitable animal models are required. Among species that could potentially be used for this purpose, nonhuman primates are particularly important, and among these the marmoset offers significant advantages. In order to demonstrate the feasibility of the application of iPSC technology to this species, here we derived lines of marmoset iPSCs. Using retroviral transduction with human Oct4, Sox2, Klf4 and c-Myc, we derived clones that fulfil critical criteria for successful reprogramming: they exhibit typical iPSC morphology; they are alkaline phosphatase positive; they express high levels of NANOG, OCT4 and SOX2 mRNAs, while the corresponding vector genes are silenced; they are immunoreactive for Oct4, TRA-1-81 and SSEA-4; and when implanted into immunodeficient mice they produce teratomas that have derivatives of all three germ layers (endoderm, alpha-fetoprotein; ectoderm, betaIII-tubulin; mesoderm, smooth muscle actin). Starting with a population of 4 x 10(5) newborn marmoset skin fibroblasts, we obtained approximately 100 colonies with iPSC-like morphology. Of these, 30 were expanded sufficiently to be cryopreserved, and, of those, 8 were characterized in more detail. These experiments provide proof of principle that iPSC technology can be adapted for use in the marmoset, as a future model of autologous cell therapy.

Type 5 17β-Hydroxysteroid Dehydrogenase (AKR1C3) Contributes to Testosterone Production in the Adrenal Reticularis

CONTEXT The human adrenal gland produces small amounts of testosterone that are increased under pathological conditions. However, the mechanisms through which the adrenal gland produces testosterone are poorly defined. OBJECTIVE Our objective was to define the role of type 5 17beta-hydroxysteroid dehydrogenase (AKR1C3) in human adrenal production of testosterone. DESIGN AND METHODS Adrenal vein sampling was used to confirm ACTH stimulation of adrenal testosterone production. Adrenal expression of AKR1C3 was studied using microarray, quantitative real-time RT-PCR, and immunohistochemical analyses. AKR1C3 knockdown was accomplished in cultured adrenal cells (H295R) using small interfering RNA, followed by measurement of testosterone production. RESULTS Acute ACTH administration significantly increased adrenal vein testosterone levels. Examination of the enzymes required for the conversion of androstenedione to testosterone using microarray analysis, quantitative real-time RT-PCR, and immunohistochemistry demonstrated that AKR1C3 was present in the adrenal gland and predominantly expressed in the zona reticularis. Decreasing adrenal cell expression of AKR1C3 mRNA and protein inhibited testosterone production in the H295R adrenal cell line. CONCLUSIONS The human adrenal gland directly secretes small, but significant, amounts of testosterone that increases in diseases of androgen excess. AKR1C3 is expressed in the human adrenal gland, with higher levels in the zona reticularis than in the zona fasciculata. AKR1C3, through its ability to convert androstenedione to testosterone, is likely responsible for adrenal testosterone production.

Dynamic assembly of chromatin complexes during cellular senescence: implications for the growth arrest of human melanocytic nevi

The retinoblastoma (RB)/p16INK4a pathway regulates senescence of human melanocytes in culture and oncogene‐induced senescence of melanocytic nevi in vivo. This senescence response is likely due to chromatin modifications because RB complexes from senescent melanocytes contain increased levels of histone deacetylase (HDAC) activity and tethered HDAC1. Here we show that HDAC1 is prominently detected in p16INK4a‐positive, senescent intradermal melanocytic nevi but not in proliferating, recurrent nevus cells that localize to the epidermal/dermal junction. To assess the role of HDAC1 in the senescence of melanocytes and nevi, we used tetracycline‐based inducible expression systems in cultured melanocytic cells. We found that HDAC1 drives a sequential and cooperative activity of chromatin remodeling effectors, including transient recruitment of Brahma (Brm1) into RB/HDAC1 mega‐complexes, formation of heterochromatin protein 1β (HP1β)/SUV39H1 foci, methylation of H3‐K9, stable association of RB with chromatin and significant global heterochromatinization. These chromatin changes coincide with expression of typical markers of senescence, including the senescent‐associated β‐galactosidase marker. Notably, formation of RB/HP1β foci and early tethering of RB to chromatin depends on intact Brm1 ATPase activity. As cells reached senescence, ejection of Brm1 from chromatin coincided with its dissociation from HP1β/RB and relocalization to protein complexes of lower molecular weight. These results provide new insights into the role of the RB pathway in regulating cellular senescence and implicate HDAC1 as a likely mediator of early chromatin remodeling events.

Stress Resistance in the Naked Mole-Rat: The Bare Essentials – A Mini-Review

Background: Studies comparing similar-sized species with disparate longevity may elucidate novel mechanisms that abrogate aging and prolong good health. We focus on the longest living rodent, the naked mole-rat. This mouse-sized mammal lives ∼8 times longer than do mice and, despite high levels of oxidative damage evident at a young age, it is not only very resistant to spontaneous neoplasia but also shows minimal decline in age-associated physiological traits. Objectives: We assess the current status of stress resistance and longevity, focusing in particular on the molecular and cellular responses to cytotoxins and other stressors between the short-lived laboratory mouse and the naked mole-rat. Results: Like other experimental animal models of lifespan extension, naked mole-rat fibroblasts are extremely tolerant of a broad spectrum of cytotoxins including heat, heavy metals, DNA-damaging agents and xenobiotics, showing LD50 values between 2- and 20-fold greater than those of fibroblasts of shorter-lived mice. Our new data reveal that naked mole-rat fibroblasts stop proliferating even at low doses of toxin whereas those mouse fibroblasts that survive treatment rapidly re-enter the cell cycle and may proliferate with DNA damage. Naked mole-rat fibroblasts also show significantly higher constitutive levels of both p53 and Nrf2 protein levels and activity, and this increases even further in response to toxins. Conclusion: Enhanced cell signaling via p53 and Nrf2 protects cells against proliferating with damage, augments clearance of damaged proteins and organelles and facilitates the maintenance of both genomic and protein integrity. These pathways collectively regulate a myriad of mechanisms which may contribute to the attenuated aging profile and sustained healthspan of the naked mole-rat. Understanding how these are regulated may be also integral to sustaining positive human healthspan well into old age and may elucidate novel therapeutics for delaying the onset and progression of physiological declines that characterize the aging process.

Senescence as an anticancer mechanism.

Senescence was originally described as a terminal nondividing state of normal human cells reached after many cell divisions in culture. The cause was shown to be shortening of telomeres, leading to telomere dysfunction and cell cycle arrest. Subsequently, a more rapid, nontelomere-dependent form of senescence, often termed stress-induced premature senescence, was described. Mostly importantly, it occurs in response to activated oncogene products. Oncogene-induced senescence has been shown to play a role in tumor suppression in vivo; it does not seem to involve changes in telomeres. A second phenomenon that plays a role in tumor suppression, which does involve progressive telomere shortening, is crisis, the state that cells reach when cell cycle checkpoints are impaired and cells can no longer respond to telomere shortening or oncogene activation by entering senescence. These two processes, oncogene-induced senescence and telomere-based crisis, exert powerful anticancer effects.

WNT4 Acts Downstream of BMP2 and Functions via β-Catenin Signaling Pathway to Regulate Human Endometrial Stromal Cell Differentiation

Differentiation of endometrial stromal cells into decidual cells is a prerequisite for successful embryo implantation. Our previous studies in the mouse have shown that bone morphogenetic protein 2 (BMP2), a morphogen belonging to the TGFβ superfamily, is essential for this differentiation process. BMP2 is markedly induced in human primary endometrial stromal cells (HESCs) as they undergo differentiation in response to steroid hormones and cAMP. The present study was undertaken to identify the BMP2-mediated molecular pathways in primary cultures of HESCs during decidualization. Using gene expression profiling, we identified wingless-related murine mammary tumor virus integration site 4 (WNT4) as a target of BMP2 regulation during decidualization. Attenuation of WNT4 expression in HESCs by small interfering RNA administration greatly reduced BMP2-induced stromal differentiation. Additionally, adenovirus-mediated overexpression of WNT4 in HESCs markedly advanced the differentiation program, indicating that it is a key regulator of decidualization. The stimulatory effect of WNT4 was accompanied by the accumulation of active β-catenin in the nuclei of decidualizing stromal cells, indicating the involvement of the canonical WNT signaling pathway. Functional inhibition of WNT4/β-catenin pathway by Dickkopf-1, an inhibitor of the canonical WNT signaling, or small interfering RNA-mediated silencing of β-catenin expression, greatly reduced the BMP2- and WNT4-induced decidualization. Gene expression profiling revealed that Forkhead box protein O1, a forkhead family transcription factor and previously reported regulator of HESC differentiation, is a common downstream mediator of both BMP2 and WNT4 signaling. Taken together, these studies uncovered a linear pathway involving BMP2, WNT4/β-catenin, and Forkhead box protein O1 that operates in human endometrium to critically control decidualization.