Person:

Calderwood, Stuart

Introduction: Ionizing radiation (IR) is a widely used approach to cancer therapy, ranking second only to surgery in rate of utilization. Responses of cancer patients to radiotherapy depend in part on the intrinsic radiosensitivity of the tumor cells. Thus, promoting tumor cell sensitivity to IR could significantly enhance the treatment outcome and quality of life for patients. Methods: Mammary tumor cells were treated by a 16-base phosphodiester-linked oligonucleotide homologous to the telomere G-rich sequence TTAGGG (T-oligo: GGTTAGGTGTAGGTTT) or a control-oligo (the partial complement, TAACCCTAACCCTAAC) followed by IR. The inhibition of tumor cell growth in vitro was assessed by cell counting and clonogenic cell survival assay. The tumorigenesis of tumor cells after various treatments was measured by tumor growth in mice. The mechanism underlying the radiosensitization by T-oligo was explored by immunofluorescent determination of phosphorylated histone H2AX ((\gamma)H2AX) foci, (\beta)-galactosidase staining, comet and Terminal deoxynucleotidyl transferase dUTP Nick End Labeling (TUNEL) assays. The efficacy of the combined treatment was assessed in a spontaneous murine mammary tumor model. Results: Pretreatment of tumor cells with T-oligo for 24 hours in vitro enhanced both senescence and apoptosis of irradiated tumor cells and reduced clonogenic potential. Radiosensitization by T-oligo was associated with increased formation and/or delayed resolution of (\gamma)H2AX DNA damage foci and fragmented DNA. T-oligo also caused radiosensitization in two in vivo mammary tumor models. Indeed, combined T-oligo and IR-treatment in vivo led to a substantial reduction in tumor growth. Of further significance, treatment with T-oligo and IR led to synergistic inhibition of the growth of spontaneous mammary carcinomas. Despite these profound antitumor properties, T-oligo and IR caused no detectable side effects under our experimental conditions. Conclusions: Pretreatment with T-oligo sensitizes mammary tumor cells to radiation in both in vitro and in vivo settings with minimal or no normal tissue side effects.

Heat shock protein (HSP)-based anticancer vaccines have undergone successful preclinical testing and are now entering clinical trial. Questions still remain, however regarding the immunological properties of HSPs. It is now accepted that many of the HSPs participate in tumor immunity, at least in part by chaperoning tumor antigenic peptides, introducing them into antigen presenting cells such as dendritic cells (DC) that display the antigens on MHC class I molecules on the cell surface and stimulate cytotoxic lymphocytes (CTL). However, in order for activated CD8+ T cells to function as effective CTL and kill tumor cells, additional signals must be induced to obtain a sturdy CTL response. These include the expression of co-stimulatory molecules on the DC surface and inflammatory events that can induce immunogenic cytokine cascades. That such events occur is indicated by the ability of Hsp70 vaccines to induce antitumor immunity and overcome tolerance to tumor antigens such as mucin1. Secondary activation of CTL can be induced by inflammatory signaling through Toll-like receptors and/or by interaction of antigen-activated T helper cells with the APC. We will discuss the role of the inflammatory properties of HSPs in tumor immunity and the potential role of HSPs in activating T helper cells and DC licensing.

Heat shock proteins (HSPs) are molecular chaperones that bind tumor antigens and mediate their uptake into antigen presenting cells. HSP–antigen complexes are then directed toward either the MHC class I pathway through antigen cross presentation or the conventional class II pathway, leading to activation of T cell subsets. Uptake of HSP-chaperoned polypeptides can involve both receptor-mediated and receptor-independent routes, and mechanisms of antigen sorting between the Class I and II pathways after uptake are currently under investigation. The processes involved in internalization of HSP–antigen complexes differ somewhat from the mechanisms previously determined for (unchaperoned) particulate and free soluble antigens. A number of studies show that HSP-facilitated antigen cross presentation requires uptake of the complexes by scavenger receptors (SR) followed by processing in the proteasome, and loading onto MHC class I molecules. In this review we have examined the roles of HSPs and SR in antigen uptake, sorting, processing, cell signaling, and activation of innate and adaptive immunity.

The target of rapamycin (TOR) is a high molecular weight protein kinase that regulates many processes in cells in response to mitogens and variations in nutrient availability. Here we have shown that mTOR in human tissue culture cells plays a key role in responses to proteotoxic stress and that reduction in mTOR levels by RNA interference leads to increase sensitivity to heat shock. This effect was accompanied by a drastic reduction in ability to synthesize heat shock proteins (HSP), including Hsp70, Hsp90 and Hsp110. As HSP transcription is regulated by heat shock transcription factor 1 (HSF1), we examined whether mTOR could directly phosphorylate this factor. Indeed, we determined that mTOR could directly phosphorylate HSF1 on serine 326, a key residue in transcriptional activation. HSF1 was phosphorylated on S326 immediately after heat shock and was triggered by other cell stressors including proteasome inhibitors and sodium arsenite. Null mutation of S326 to alanine led to loss of ability to activate an HSF1-regulated promoter-reporter construct, indicating a direct role for mTOR and S326 in transcriptional regulation of HSP genes during stress. As mTOR is known to exist in at least two intracellular complexes, mTORC1 and mTOR2 we examined which complex might interact with HSF1. Indeed mTORC1 inhibitor rapamycin prevented HSF1-S326 phosphorylation, suggesting that this complex is involved in HSF1 regulation in stress. Our experiments therefore suggest a key role for mTORC1 in transcriptional responses to proteotoxic stress.

Introduction: It is still uncertain whether metastasis is predominantly an early or late event in tumor progression. The detection of early metastases and cells responsible for the dissemination may therefore have significant clinical implications. Methods: Lung dissemination and/or metastasis were investigated in mice carrying the polyomavirus middle-T oncogene (PyMT) during different stages of mammary tumorigenesis using the colony forming assay. Immunocytochemical or immunohistochemical staining was used to identify subpopulations of cells responsible for lung dissemination and metastasis. Histological examination was used to show primary and metastatic tumors. The tumor-initiating and metastatic capacity of cells expressing stem cell markers was assessed in syngeneic wild-type (WT) mice whose mammary fat pads were injected with these cells. Results: Metastatic mammary epithelial cells were detected in the lungs of mice carrying the PyMT oncogene (MMT mice). These cells were observed early in breast tumorigenesis when the mammary tree appeared by histological inspection to be normal (or at a premalignant stage), suggesting the possession of disseminating and metastatic capacity even before full malignant transformation. Some of the disseminated cells and lung metastases displayed surface stem cell markers. These findings suggest that stem cells from apparently precancerous primary lesions could be a source of metastasis. Indeed, injection of lung tissue cells from MMT mice into syngeneic WT mice resulted in the formation of mammary tumors. These tumors resembled their parent mammary tumors in the MMT donors as well as grafted tumors derived from mammary tumor cells. Furthermore, when we injected lung tissue cells from GFP MMT mice into the fat pads of recipient WT mice, disseminated or metastatic GFP-expressing cells were detected in the lungs, lymph nodes and blood of the recipient WT mice. We finally identified a subpopulation of mammary epithelial/tumor cells expressing CD44 and Sca1 that was largely responsible for dissemination and metastasis in MMT mice. Conclusions: The tumorigenic and metastatic potential of a subpopulation of mammary epithelial/tumor cells in MMT mice is endowed relatively early in mammary neoplasms and suggests a potential role for cancer stem cell sub-populations in metastasis.

Background: Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Principal Findings: Pretreatment of hydrogen peroxide ((H_2O_2)) specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. (H_2O_2) exposure impaired the HSP40/HSP70 induction as heat shock response (HSR) and the unfolded protein recovery, and enhanced (eIF2\alpha) phosphorylation and/or XBP1 splicing, land marks of ER stress. These (H_2O_2)-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked (H_2O_2)–mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for (H_2O_2)–mediated enhanced heat sensitivity. Conclusions: (H_2O_2) blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.

MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.

Heat shock factor 1 (HSF1) regulates one of the major pathways of protein quality control and is essential for deterrence of protein-folding disorders, particularly in neuronal cells. However, HSF1 activity declines with age, a change that may open the door to progression of neurodegenerative disorders such as Huntington's disease. We have investigated mechanisms of HSF1 regulation that may become compromised with age. HSF1 binds stably to the catalytic domain of protein kinase A (PKAcα) and becomes phosphorylated on at least one regulatory serine residue (S320). We show here that PKA is essential for effective transcription of HSP genes by HSF1. PKA triggers a cascade involving HSF1 binding to the histone acetylase p300 and positive translation elongation factor 1 (p-TEFb) and phosphorylation of the c-terminal domain of RNA polymerase II, a key mechanism in the downstream steps of HSF1-mediated transcription. This cascade appears to play a key role in protein quality control in neuronal cells expressing aggregation-prone proteins with long poly-glutamine (poly-Q) tracts. Such proteins formed inclusion bodies that could be resolved by HSF1 activation during heat shock. Resolution of the inclusions was inhibited by knockdown of HSF1, PKAcα, or the pTEFb component CDK9, indicating a key role for the HSF1-PKA cascade in protein quality control.

Scavenger receptor expressed by endothelial cell-I (SREC-I) is a class F scavenger receptor expressed by immune cells with a significant role in CD8+- and CD4+-mediated T cell immunity. This receptor can also modulate the function of toll-like receptors (TLRs), which play essential roles in innate immunity. Earlier, it was found that human monocyte/macrophage THP1 cells and bone marrow-derived macrophages from mice exhibited increased responses to polyinosine–polycytidylic acid (poly I:C, PIC) and CpG (unmethylated) DNA and enhanced production of inflammatory cytokines with overexpressed SREC-I. Our data also showed that intracellular/endocytic TLR3 and TLR9 could directly interact with SREC-I in the presence of their respective ligands. We also observed that the internalized ligand along with TLR3/TLR9 colocalized in the endosome in macrophages and THP-1 cells overexpressing these receptors. In the absence of these ligands, there was no detectable colocalization between the SREC-I and endocytic TLRs. Earlier, it was shown that SREC-I stimulated double-stranded RNA/CpGDNA-mediated TLR3/TLR9 activation of the innate immune response by triggering signaling through the NF-κB, IRF3, and MAP kinase pathways leading to transcription of cytokine genes. We also established that SREC-I can associate with plasma membrane TLRs, such as TLR2 and TLR4. We demonstrated that SREC-I–TLR4 signals more efficiently from lipid microdomain in which lipopolysaccharide (LPS) can associate with SREC-I–TLR4 complex. We also proved that SREC-I is an alternate receptor for LPS capable of internalizing the complex and for endocytic TLR ligands as well. This binding activated endocytic TLR-mediated downstream cytokine production in THP1 cells and macrophages. Finally, SREC-I could also form complexes with TLR2 and induce the release of cytokines in the presence of bacterial, viral, and fungal ligands.

TRIM28 is a multidomain protein with versatile functions in transcription and DNA repair. Recently it was shown that this factor plays unanticipated roles in transcriptional elongation. TRIM28 was shown to stabilize the pausing of RNA polymerase II (Pol II) close to the transcriptional start site in many unactivated genes, permitting Pol II accumulation and readying genes for induction. In addition, the factor was shown to respond rapidly to signals accompanying transcriptional activation permitting the productive elongation of RNA by previously paused Pol II. We discuss here critical regulatory mechanisms of TRIM28 in transcriptional control and DNA repair that may illuminate the novel roles of this factor in pausing and elongation of Pol II.