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Purpose: Klotho is a transmembrane protein which can be shed, act as a circulating hormone and modulate the insulin-like growth factor (IGF)-I and the fibroblast growth factor (FGF) pathways. We have recently identified klotho as a tumor suppressor in breast cancer. Klotho is expressed in the normal pancreas and both the IGF-I and FGF pathways are involved in pancreatic cancer development. We, therefore, undertook to study the expression and activity of klotho in pancreatic cancer.Experimental Design: Klotho expression was studied using immunohistochemistry and quantitative RT-PCR. Effects of klotho on cell growth were assessed in the pancreatic cancer cells Panc1, MiaPaCa2, and Colo357, using colony and MTT assays and xenograft models. Signaling pathway activity was measured by Western blotting.Results: Klotho expression is downregulated in pancreatic adenocarcinoma. Overexpression of klotho, or treatment with soluble klotho, reduced growth of pancreatic cancer cells in vitro and in vivo, and inhibited activation of the IGF-I and the bFGF pathways. KL1 is a klotho subdomain formed by cleavage or alternative splicing. Compared with the full-length protein, KL1 showed similar growth inhibitory activity but did not promote FGF23 signaling. Thus, its administration to mice showed favorable safety profile.Conclusions: These studies indicate klotho as a potential tumor suppressor in pancreatic cancer, and suggest, for the first time, that klotho tumor suppressive activities are mediated through its KL1 domain. These results suggest the use of klotho or KL1 as potential strategy for the development of novel therapeutic interventions for pancreatic cancer. Clin Cancer Res; 17(13); 4254–66. ©2011 AACR.

Incidence of spontaneous micrometastases

Supplementary Figure S1: Spontaneous brain macrometastases analyzed by MRI or histology

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood–brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359–71. ©2016 AACR.

Supplementary Methods

Supplementary Figures S1-S2; Supplementary Tables S1-S2.

Incidence of spontaneous brain macrometastases

Supplementary figure legends

The major cause of melanoma mortality is metastasis to distant organs, including lungs and brain. Reciprocal interactions of metastasizing tumor cells with stromal cells in secondary sites play a critical role in all stages of tumorigenesis and metastasis. Changes in the metastatic microenvironment were shown to precede clinically relevant metastases, and may occur prior to the arrival of disseminated tumor cells to the distant organ, thus creating a hospitable "premetastatic niche." Exosomes secreted by tumor cells were demonstrated to play an important role in the preparation of a hospitable metastatic niche. However, the functional role of melanoma-derived exosomes on metastatic niche formation, and the downstream pathways activated in stromal cells at the metastatic niche are largely unresolved. Here we show that extracellular vesicles (EVs) secreted by metastatic melanoma cells that spontaneously metastasize to lungs and to brain, activate proinflammatory signaling in lung fibroblasts and in astrocytes. Interestingly, unlike paracrine signaling by melanoma cells, EVs secreted by metastatic melanoma cells instigated a proinflammatory gene signature in lung fibroblasts but did not activate wound-healing functions, suggesting that tumor cell-secreted EVs activate distinct CAF characteristics and tumor-promoting functions. Moreover, melanoma-secreted EVs also activated proinflammatory signaling in astrocytes, indicating that EV-mediated reprogramming of stromal cells is a general mechanism of modulating the metastatic niche in multiple distant organs. Thus, our study demonstrates that melanoma-derived EVs reprogram tumor-promoting functions in stromal cells in a distinct manner, implicating a central role for tumor-derived EV signaling in promoting the formation of an inflammatory metastatic niche.

Adrenergic signaling plays a central role in physiological regulation, including modulation of processes in the immune system. However, the effects of adrenergic activation on antibody-mediated immune response remain unknown. Here, we investigate the effects of stress - induced β-adrenergic receptor activation on the B cell response at molecular and the systemic levels. We find that β-adrenergic agonist treatment of B cells from three convalescent SARS coronavirus-2 donors, reduced both membrane IgG expression and clonal expansion when the cells were stimulated ex vivo with spike receptor binding domain (RBD). Interestingly, monoclonal anti-RBD antibodies cloned from B cells cultured in the presence of β-adrenergic agonist, exhibited higher affinity for RBD compared to antibodies cloned from control cultures, suggesting that clones under stress exhibit higher antigen affinity. As a corollary, following ovalbumin immunization in mice, physiological stress during germinal center reaction phase increased the levels of antigen specific serum IgG. At the same time, B cell clonal expansion and membrane IgG expression were reduced, along with an increase in MHC class II expression. These effects were abolished by treatment with the non-selective β-adrenergic antagonist, propranolol. Our study suggests that under stress conditions selection of high affinity variants comes in the expense of clonal expansion.

Evidence has emerged that some healthy individuals with a history of exposure to Mycobacterium tuberculosis (Mtb) can develop protective antibody responses. However, it is not known whether patients with active tuberculosis elicit protective antibodies, and if they do, which bacterial antigens are targeted. To investigate the B cell responses during active infection, we generated a panel of monoclonal antibodies isolated from memory B cells of one patient. The antibodies, members of four distinct B cell clones, were directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4–36 and p4–163 from two different B cell clones showed protective efficacy against Mtb and Mycobacterium bovis-BCG in an ex vivo human whole blood growth inhibition assay. Germline versions of p4–36 and p4–163 could no longer bind Mtb, implying that affinity maturation was vital for their activity. Crystal structures of p4–36 and a closely related clonal variant of p4–163, p4–170, complexed to PstS1 were determined at a resolution of 2.1Å and 2.4Å and revealed that the two antibodies recognize two distinctive epitopes on PstS1. As a proof of principle, p4–36 and p4–163 were used in a passive vaccination setting in aerosol Mtb-infected Balb/c mice, where both antibodies reduced bacterial lung burden by 50% after a single injection prior to Mtb infection. Our study shows that inhibitory B cell responses arise during active tuberculosis and identifies PstS1 as a target for elicitation of anti-Mtb antibodies.

Mycobacterium tuberculosis (Mtb) exposure drives antibody responses, but whether patients with active tuberculosis elicit protective antibodies, and against which antigens, is still unclear. Here we generate monoclonal antibodies from memory B cells of one patient to investigate the B cell responses during active infection. The antibodies, members of four distinct B cell clones, are directed against the Mtb phosphate transporter subunit PstS1. Antibodies p4-36 and p4-163 reduce Mycobacterium bovis-BCG and Mtb levels in an ex vivo human whole blood growth inhibition assay in an FcR-dependent manner; meanwhile, germline versions of p4-36 and p4-163 do not bind Mtb. Crystal structures of p4-36 and p4-170, complexed to PstS1, are determined at 2.1 Å and 2.4 Å resolution, respectively, to reveal two distinctive PstS1 epitopes. Lastly, a prophylactic p4-36 and p4-163 treatment in Mtb-infected Balb/c mice reduces bacterial lung burden by 50%. Our study shows that inhibitory anti-PstS1 B cell responses arise during active tuberculosis., Antibody responses against Mycobacteria infection have been reported, but whether and how they impact anti-bacteria immunity in the host is unclear. Here the authors characterize human anti-Mycobacteria antibodies to find them targeting a Mycobacteria transporter protein, PstS1, show distinct interaction modes in crystal structure, and mediate protection in vitro.

Cancer-associated fibroblasts (CAFs) are highly prominent in breast tumors, but their functional heterogeneity and origin are still largely unresolved. We report that bone marrow (BM)–derived mesenchymal stromal cells (MSCs) are recruited to primary breast tumors and to lung metastases and differentiate to a distinct subpopulation of CAFs. We show that BM-derived CAFs are functionally important for tumor growth and enhance angiogenesis via up-regulation of Clusterin. Using newly generated transgenic mice and adoptive BM transplantations, we demonstrate that BM-derived fibroblasts are a substantial source of CAFs in the tumor microenvironment. Unlike resident CAFs, BM-derived CAFs do not express PDGFRα, and their recruitment resulted in a decrease in the percentage of PDGFRα-expressing CAFs. Strikingly, decrease in PDGFRα in breast cancer patients was associated with worse prognosis, suggesting that BM-derived CAFs may have deleterious effects on survival. Therefore, PDGFRα expression distinguishes two functionally unique CAF populations in breast tumors and metastases and may have important implications for patient stratification and precision therapeutics.

The treatment of neurodegenerative diseases remains a tremendous challenge due to the limited access of molecules across the blood–brain barrier, especially large molecules such as peptides and proteins. As a result, at most, a small percentage of a drug that is administered systemically will reach the central nervous system in its active form. Currently, research in the field focuses on developing safer and more effective approaches to deliver peptides and proteins into the central nervous system. Multiple strategies have been developed for this purpose. However, noninvasive approaches, such as nanostructured protein delivery carriers and intranasal administration, seem to be the most promising strategies for the treatment of chronic diseases, which require long-term interventions. These approaches are both target-specific and able to rapidly bypass the blood–brain barrier. In this Perspective, we detail some of these strategies and discuss some of the potential pitfalls and opportunities in this field. ...

Malignant melanoma is the deadliest of skin cancers. Melanoma frequently metastasizes to the brain, resulting in dismal survival. Nevertheless, mechanisms that govern early metastatic growth and the interactions of disseminated metastatic cells with the brain microenvironment are largely unknown. To study the hallmarks of brain metastatic niche formation, we established a transplantable model of spontaneous melanoma brain metastasis in immunocompetent mice and developed molecular tools for quantitative detection of brain micrometastases. Here we demonstrate that micrometastases are associated with instigation of astrogliosis, neuroinflammation, and hyperpermeability of the blood–brain barrier. Furthermore, we show a functional role for astrocytes in facilitating initial growth of melanoma cells. Our findings suggest that astrogliosis, physiologically instigated as a brain tissue damage response, is hijacked by tumor cells to support metastatic growth. Studying spontaneous melanoma brain metastasis in a clinically relevant setting is the key to developing therapeutic approaches that may prevent brain metastatic relapse. Cancer Res; 76(15); 4359–71. ©2016 AACR.

Purpose: Klotho is a transmembrane protein which can be shed, act as a circulating hormone and modulate the insulin-like growth factor (IGF)-I and the fibroblast growth factor (FGF) pathways. We have recently identified klotho as a tumor suppressor in breast cancer. Klotho is expressed in the normal pancreas and both the IGF-I and FGF pathways are involved in pancreatic cancer development. We, therefore, undertook to study the expression and activity of klotho in pancreatic cancer. Experimental Design: Klotho expression was studied using immunohistochemistry and quantitative RT-PCR. Effects of klotho on cell growth were assessed in the pancreatic cancer cells Panc1, MiaPaCa2, and Colo357, using colony and MTT assays and xenograft models. Signaling pathway activity was measured by Western blotting. Results: Klotho expression is downregulated in pancreatic adenocarcinoma. Overexpression of klotho, or treatment with soluble klotho, reduced growth of pancreatic cancer cells in vitro and in vivo, and inhibited activation of the IGF-I and the bFGF pathways. KL1 is a klotho subdomain formed by cleavage or alternative splicing. Compared with the full-length protein, KL1 showed similar growth inhibitory activity but did not promote FGF23 signaling. Thus, its administration to mice showed favorable safety profile. Conclusions: These studies indicate klotho as a potential tumor suppressor in pancreatic cancer, and suggest, for the first time, that klotho tumor suppressive activities are mediated through its KL1 domain. These results suggest the use of klotho or KL1 as potential strategy for the development of novel therapeutic interventions for pancreatic cancer. Clin Cancer Res; 17(13); 4254–66. ©2011 AACR.

Klotho is a transmembrane protein that can be shed and act as a circulating hormone and is a putative tumor suppressor in breast cancer. A functional variant of KLOTHO (KL-VS) contains two amino acid substitutions F352V and C370S and shows reduced activity. Germ-line mutations in BRCA1 and BRCA2 substantially increase lifetime risk of breast and ovarian cancers. Yet, penetrance of deleterious BRCA1 and BRCA2 mutations is incomplete even among carriers of identical mutations. We examined the association between KL-VS and cancer risk among 1115 Ashkenazi Jewish women: 236 non-carriers, 631 BRCA1 (185delAG, 5382insC) carriers and 248 BRCA2 (6174delT) carriers. Among BRCA1 carriers, heterozygosity for the KL-VS allele was associated with increased breast and ovarian cancer risk (hazard ratio 1.40, 95% confidence intervals 1.08-1.83, P=0.01) and younger age at breast cancer diagnosis (median age 48 vs 43 P=0.04). KLOTHO and BRCA2 are located on 13q12, and we identified linkage disequilibrium between KL-VS and BRCA2 6174delT mutation. Studies in breast cancer cells showed reduced growth inhibitory activity and reduced secretion of klotho F352V compared with wild-type klotho. These data suggest KL-VS as a breast and ovarian cancer risk modifier among BRCA1 mutation carriers. If validated in additional cohorts, the presence of KL-VS may serve as a predictor of cancer risk among BRCA1 mutation carriers.

Studies have conjectured that nonsteroidal anti-inflammatory drugs (NSAID) inhibit growth of various malignancies by inhibiting cyclooxygenase-2 (COX-2) enzyme activity. Yet, several lines of evidence indicate that a COX-2–independent mechanism may also be involved in their antitumor effects. Here, we report that NSAIDs may inhibit the growth of glioblastoma multiforme (GBM) cells through COX-2–independent mechanisms, including up-regulation of both 15-hydroxyprostaglandin dehydrogenase (15-PGDH, the key prostaglandin catabolic enzyme) and the cell cycle inhibitor p21. Using Western blot and real-time PCR analysis in various GBM cell lines, we observed up-regulation of 15-PGDH and p21 after NSAIDs treatment. To elucidate the role of 15-PGDH in GBM, transfection assays were conducted using the T98G GBM cell line. Overexpression of 15-PGDH suppressed cell growth and was associated with increased expression of p21. In an attempt to investigate the roles of COX-2, 15-PGDH, and p21 in the inhibition of growth of GBM, small interfering RNA (siRNA) against each of these proteins was transfected into T98G cells. Inhibition of growth mediated by NSAIDs was partially reversed after knockdown of either 15-PGDH or p21, but not after COX-2 knockdown. Moreover, expression level of p21 was not affected in COX-2 siRNA transfected cells. Our studies provide evidence that the up-regulation of 15-PGDH induced by NSAIDs has the potential to inhibit growth of GBM, in part, by up-regulation of p21 possibly independent from COX-2 enzymatic function. [Cancer Res 2008;68(17):6978–86]

Restriction of glutamine synthetase to the nervous system is mainly achieved through the mutual function of the glucocorticoid receptor and the neural restrictive silencing factor, NRSF/REST. Glucocorticoids induce glutamine synthetase expression in neural tissues while NRSF/REST represses the hormonal response in non-neural cells. NRSF/REST is a modular protein that contains two independent repression domains, at the N and C termini of the molecule, and is dominantly expressed in nonneural cells. Neural tissues express however splice variants, REST4/5, which contain the repression domain at the N, but not at the C terminus of the molecule. Here we show that full-length NRSF/REST or its C-terminal domain can inhibit almost completely the induction of gene transcription by glucocorticoids. By contrast, the N-terminal domain not only fails to repress the hormonal response but rather stimulates it markedly. The inductive activity of the N-terminal domain is mediated by hBrm, which is recruited to the promoter only in the concomitant presence of GR. Importantly, a similar inductive activity is also exerted by the splice variant REST4. These findings raise the possibility that NRSF/REST exhibits a dual role in regulation of glutamine synthetase. It represses gene induction in nonneural cells and enhances the hormonal response, via its splice variant, in the nervous system.

Internal initiation of translation, whereby ribosomes are directed to internal AUG codon independently of the 5′ end of the mRNA, has been observed rarely in higher eucaryotes and has not been demonstrated in living yeast. We report here that starved yeast cells are capable of initiating translation of a dicistronic message internally. The studied element that functions as an internal ribosome entry site (IRES) is hardly functional or not functional at all in logarithmically growing cells. Moreover, during the logarithmic growth phase, this element seems to inhibit translation reinitiation when placed as an intercistronic spacer or to inhibit translation when placed in the 5′-untranslated region of a monocistronic message. Inhibition of translation is likely due to the putative strong secondary structure of the IRES that interferes with the cap-dependent scanning process. When cells exit the logarithmic growth phase, or when artificially starved for carbon source, translation of the IRES-containing messages is substantially induced. Our findings imply that the capacity to translate internally is a characteristic of starved rather than vegetatively growing yeast cells.

Klotho is a transmembranal protein, which can be cleaved, shed and act as a circulating hormone. Klotho deficient mice present a syndrome of early aging, while overexpression of klotho extends lifespan. Klotho is a potent inhibitor of the insulin growth factor (IGF)-1 and the basic fibroblast growth factor (FGF) pathways. Klotho is also an essential co-factor for the activity of FGF23 and thus serves as a major regulator of phosphate homeostasis. We have recently identified klotho as a tumor suppressor in breast cancer. As klotho is expressed in the normal pancreas and as the IGF-1 and FGF pathways are involved in pancreatic cancer development, we undertook to study the expression and activity of klotho in pancreatic cancer. Immunohistochemistry analysis revealed significantly reduced expression of klotho in pancreatic cancer compared to normal pancreas, and low levels of klotho mRNA were noted in the pancreatic cancer cells Panc1, Colo357 and MiaPaCa2 compared to normal pancreas cells. Further studies revealed that klotho silencing is mediated by epigenetic mechanisms. Overexpression of klotho or treatment with soluble klotho (8pM) reduced clonal growth of pancreatic cancer cell lines, and a synergistic effect was observed in combination with chemotherapeutic drugs. Daily injections of klotho (10μg/kg) inhibited tumor formation of Panc1 cells in athymic mice. Klotho inhibited activation of the IGF-1 pathway in pancreatic cancer cells. As klotho further enhanced growth inhibition by the IGF-1R inhibitor AG1024, modulation of additional signaling pathways by klotho was suspected. Indeed, klotho overexpression inhibited the bFGF pathway in these cells. The extracellular region of klotho is composed of two internal repeats, KL1 and KL2, and each of them can be cleaved and act separately. While both domains share homology to glycosidase, a structural model suggested that only KL1 retains enzymatic activity. While the KL2 domain did not show growth inhibitory activities, KL1 effectively slowed growth of pancreatic cancer cells, both in vitro and in vivo and was found to be a potent inhibitor of the IGF-1 and bFGF pathways. Importantly, KL1 did not promote FGF23 signaling and therefore, in contrary to the full-length protein, its administration to mice did not affect phosphate homeostasis. In conclusion, these studies indicate klotho as a potent tumor suppressor in pancreatic cancer and suggest, for the first time, that klotho growth inhibitory activities are mediated by the KL1 domain, possibly due to its putative enzymatic activities. As the KL1 domain is not involved in regulation of phosphate homeostasis, its administration may serve as a potential safe and effective novel strategy for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2192. doi:10.1158/1538-7445.AM2011-2192

Cancer-Associated Fibroblasts (CAFs) are the most prominent stromal cell type in breast tumors. CAFs promote tumor growth and metastasis by multiple mechanisms, including by mediating tumor-promoting inflammation. Immune modulation in the tumor microenvironment plays a central role in determining disease outcome. However, the functional interactions of CAFs with immune cells are largely unknown. Here we report a novel signaling axis between fibroblasts, cancer cells and immune cells in breast tumors that drives an immunosuppressive microenvironment, mediated by CAF-derived Chi3L1. We demonstrate that Chi3L1 is highly upregulated in CAFs isolated from mammary tumors and pulmonary metastases of transgenic mice, and in the stroma of human breast carcinomas. Genetic ablation of Chi3L1 in fibroblasts in vivo attenuated tumor growth, macrophage recruitment and reprogramming to an M2-like phenotype, enhanced tumor infiltration by CD8+ and CD4+ T cells and promoted a Th1 phenotype. These results indicate that CAF-derived Chi3L1 promotes tumor growth and shifts the balance of the immune milieu towards type 2 immunity. Taken together, our findings implicate fibroblast-derived Chi3L1 as a novel key player in the complex reciprocal interactions of stromal cells that facilitate tumor progression and metastasis, and suggest that targeting Chi3L1 may be clinically beneficial in breast cancer.