Detailed protocol for optimised expression and purification of functional monomeric human Heat Shock Factor 1.

Heat Shock Factor 1 (HSF1) is the master regulator of the heat shock response, a universal survival mechanism throughout eukaryotic species used to buffer potentially lethal proteotoxic conditions. HSF1's function in vivo is regulated by several factors, including post translational modifications and elevated temperatures, whereupon it forms trimers to bind with heat shock elements in DNA. Unsurprisingly, HSF1 is also extremely sensitive to elevated temperatures in vitro, which poses specific technical challenges when producing HSF1 using a recombinant expression system. Although there are several useful publications which outline steps taken for HSF1 expression and purification, studies that describe specific strategies and detailed protocols to overcome HSF1 trimerisation and degradation are currently lacking. Herein, we have reported our detailed experimental protocol for the expression and purification of monomeric human HSF1 (HsHSF1) as a major species. We also propose a refined method of inducing HsHSF1 activation in vitro, that we consider more accurately mimics HsHSF1 activation in vivo and is therefore more physiologically relevant.

PtdIns(3,4,5)P3-dependent Rac Exchanger 1 (PREX1) Rac-Guanine Nucleotide Exchange Factor (GEF) Activity Promotes Breast Cancer Cell Proliferation and Tumor Growth via Activation of Extracellular Signal-regulated Kinase 1/2 (ERK1/2) Signaling.

PtdIns(3,4,5)P3-dependent Rac exchanger 1 (PREX1) is a Rac-guanine nucleotide exchange factor (GEF) overexpressed in a significant proportion of human breast cancers that integrates signals from upstream ErbB2/3 and CXCR4 membrane surface receptors. However, the PREX1 domains that facilitate its oncogenic activity and downstream signaling are not completely understood. We identify that ERK1/2 MAPK acts downstream of PREX1 and contributes to PREX1-mediated anchorage-independent cell growth. PREX1 overexpression increased but its shRNA knockdown decreased ERK1/2 phosphorylation in response to EGF/IGF-1 stimulation, resulting in induction of the cell cycle regulators cyclin D1 and p21(WAF1/CIP1) PREX1-mediated ERK1/2 phosphorylation, anchorage-independent cell growth, and cell migration were suppressed by inhibition of MEK1/2/ERK1/2 signaling. PREX1 overexpression reduced staurosporine-induced apoptosis whereas its shRNA knockdown promoted apoptosis in response to staurosporine or the anti-estrogen drug tamoxifen. Expression of wild-type but not GEF-inactive PREX1 increased anchorage-independent cell growth. In addition, mouse xenograft studies revealed that expression of wild-type but not GEF-dead PREX1 resulted in the formation of larger tumors that displayed increased phosphorylation of ERK1/2 but not AKT. The impaired anchorage-independent cell growth, apoptosis, and ERK1/2 signaling observed in stable PREX1 knockdown cells was restored by expression of wild-type but not GEF-dead-PREX1. Therefore, PREX1-Rac-GEF activity is critical for PREX1-dependent anchorage-independent cell growth and xenograft tumor growth and may represent a possible therapeutic target for breast cancers that exhibit PREX1 overexpression.

Mammographically dense human breast tissue stimulates MCF10DCIS.com progression to invasive lesions and metastasis.

BACKGROUND: High mammographic density (HMD) not only confers a significantly increased risk of breast cancer (BC) but also is associated with BCs of more advanced stages. However, it is unclear whether BC progression and metastasis are stimulated by HMD. We investigated whether patient-derived HMD breast tissue could stimulate the progression of MCF10DCIS.com cells compared with patient-matched low mammographic density (LMD) tissue. METHODS: Sterile breast specimens were obtained immediately after prophylactic mastectomy from high-risk women (n = 10). HMD and LMD regions of each specimen were resected under radiological guidance. Human MCF10DCIS.com cells, a model of ductal carcinoma in situ (DCIS), were implanted into silicone biochambers in the groins of severe combined immunodeficiency mice, either alone or with matched LMD or HMD tissue (1:1), and maintained for 6 weeks. We assessed biochamber weight as a measure of primary tumour growth, histological grade of the biochamber material, circulating tumour cells and metastatic burden by luciferase and histology. All statistical tests were two-sided. RESULTS: HMD breast tissue led to increased primary tumour take, increased biochamber weight and increased proportions of high-grade DCIS and grade 3 invasive BCs compared with LMD. This correlated with an increased metastatic burden in the mice co-implanted with HMD tissue. CONCLUSIONS: Our study is the first to explore the direct effect of HMD and LMD human breast tissue on the progression and dissemination of BC cells in vivo. The results suggest that HMD status should be a consideration in decision-making for management of patients with DCIS lesions.

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Context-dependent role of Grb7 in HER2+ve and triple-negative breast cancer cell lines.

Grb7 is an adapter protein, aberrantly co-overexpressed with HER2 and identified as an independent prognostic marker in breast cancer. It has been established that Grb7 exacerbates the cellular growth and migratory behaviour of HER2+ve breast cancer cells. Less is known about Grb7's role in the context of HER2-ve cells. Here we directly compare the effect of stable Grb7 knockdown in oestrogen sensitive (T47D), HER2+ve (SKBR3) and triple-negative (MDA-MB-468 and MDA-MB-231) breast cancer cell lines on anchorage dependent and independent cell growth, wound healing and chemotaxis. All cell lines showed reduced ability to migrate upon Grb7 knockdown, despite their greatly varied endogenous levels of Grb7. Decreased cell proliferation was not observed in any of the cell lines upon Grb7 knockdown; however, decreased ability to form colonies was observed for all but the oestrogen sensitive cell line, depending upon the stringency of the growth conditions. The data reveal that Grb7 plays an important role in breast cancer progression, beyond the context of HER2+ve cell types.

Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status.

HSF1 (heat-shock factor 1) is the master regulator of the heat-shock response; however, it is also activated by cancer-associated stresses and supports cellular transformation and cancer progression. We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53-dependent mechanism. To examine this relationship more specifically, we ectopically co-expressed mutant p53(R273H) and HSF1 in the human mammary epithelial cell line MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, leaving mutant p53(R273H) expression intact, HSF1 was observed to greatly enhance clonogenic growth of the cells, indicating that HSF1 suppressed clonogenicity via wild-type p53. To confirm this we ectopically expressed HSF1 in non-transformed and H-Ras(V12)-transformed MCF10A cells. As expected, HSF1 significantly reduced clonogenicity, altering wild-type p53 target gene expression levels consistent with a role of HSF1 increasing wild-type p53 activity. In support of this finding, knockdown of wild-type p53 negated the inhibitory effects of HSF1 expression. We thus show that HSF1 can affect clonogenic growth in a p53 context-dependent manner, and can act via both mutant and wild-type p53 to bring about divergent effects upon clonogenicity. These findings have important implications for our understanding of HSF1's divergent roles in cancer cell growth and survival as well as its disparate effect on mutant and wild-type p53.

HSP90 inhibitors enhance differentiation and MITF (microphthalmia transcription factor) activity in osteoclast progenitors.

The HSP90 (heat-shock protein 90) inhibitor 17-AAG (17-allylamino-demethoxygeldanamycin) increases osteoclast formation both in vitro and in vivo, an action that can enhance cancer invasion and growth in the bone microenvironment. The cellular mechanisms through which 17-AAG exerts this action are not understood. Thus we sought to clarify the actions of 17-AAG on osteoclasts and determine whether other HSP90 inhibitors had similar properties. We determined that 17-AAG and the structurally unrelated HSP90 inhibitors CCT018159 and NVP-AUY922 dose-dependently increased RANKL [receptor activator of NF-κB (nuclear factor κB) ligand]-stimulated osteoclastogenesis in mouse bone marrow and pre-osteoclastic RAW264.7 cell cultures. Moreover, 17-AAG also enhanced RANKL- and TNF (tumour necrosis factor)-elicited osteoclastogenesis, but did not affect RANKL-induced osteoclast survival, suggesting that only differentiation mechanisms are targeted. 17-AAG affected the later stages of progenitor maturation (after 3 days of incubation), whereas the osteoclast formation enhancer TGFß (transforming growth factor ß) acted prior to this, suggesting different mechanisms of action. In studies of RANKL-elicited intracellular signalling, 17-AAG treatment did not increase c-Fos or NFAT (nuclear factor of activated T-cells) c1 protein levels nor did 17-AAG increase activity in luciferase-based NF-κB- and NFAT-response assays. In contrast, 17-AAG treatment (and RANKL treatment) increased both MITF (microphthalmia-associated transcription factor) protein levels and MITF-dependent vATPase-d2 (V-type proton ATPase subunit d2) gene promoter activity. These results indicate that HSP90 inhibitors enhance osteoclast differentiation in an NFATc1-independent manner that involves elevated MITF levels and activity.

Inositol polyphosphate 4-phosphatase II regulates PI3K/Akt signaling and is lost in human basal-like breast cancers.

Inositol polyphosphate 4-phosphatase-II (INPP4B) is a regulator of the phosphoinositide 3-kinase (PI3K) signaling pathway and is implicated as a tumor suppressor in epithelial carcinomas. INPP4B loss of heterozygosity (LOH) is detected in some human breast cancers; however, the expression of INPP4B protein in breast cancer subtypes and the normal breast is unknown. We report here that INPP4B is expressed in nonproliferative estrogen receptor (ER)-positive cells in the normal breast, and in ER-positive, but not negative, breast cancer cell lines. INPP4B knockdown in ER-positive breast cancer cells increased Akt activation, cell proliferation, and xenograft tumor growth. Conversely, reconstitution of INPP4B expression in ER-negative, INPP4B-null human breast cancer cells reduced Akt activation and anchorage-independent growth. INPP4B protein expression was frequently lost in primary human breast carcinomas, associated with high clinical grade and tumor size and loss of hormone receptors and was lost most commonly in aggressive basal-like breast carcinomas. INPP4B protein loss was also frequently observed in phosphatase and tensin homolog (PTEN)-null tumors. These studies provide evidence that INPP4B functions as a tumor suppressor by negatively regulating normal and malignant mammary epithelial cell proliferation through regulation of the PI3K/Akt signaling pathway, and that loss of INPP4B protein is a marker of aggressive basal-like breast carcinomas.

Inhibition of autophagy; an opportunity for the treatment of cancer resistance.

The process of macroautophagy plays a pivotal role in the degradation of long-lived, superfluous, and damaged proteins and organelles, which are later recycled for cellular use. Normal cells rely on autophagy to combat various stressors and insults to ensure survival. However, autophagy is often upregulated in cancer cells, promoting a more aggressive phenotype that allows mutated cells to evade death after exposure to therapeutic treatments. As a result, autophagy has emerged as a significant factor in therapeutic resistance across many cancer types, with underlying mechanisms such as DNA damage, cell cycle arrest, and immune evasion. This review provides a comprehensive summary of the role of autophagy in therapeutic resistance and the limitations of available autophagic inhibitors in cancer treatment. It also highlights the urgent need to explore new inhibitors that can synergize with existing therapies to achieve better patient treatment outcomes. Advancing research in this field is crucial for developing more effective treatments that can help improve the lives of cancer patients.

FKBP25 regulates myoblast viability and migration and is differentially expressed in in vivo models of muscle adaptation.

FKBP25 (FKBP3 gene) is a dual-domain PPIase protein that consists of a C-terminal PPIase domain and an N-terminal basic tilted helix bundle (BTHB). The PPIase domain of FKBP25 has been shown to bind to microtubules, which has impacts upon microtubule polymerisation and cell cycle progression. Using quantitative proteomics, it was recently found that FKBP25 was expressed in the top 10% of the mouse skeletal muscle proteome. However, to date there have been few studies investigating the role of FKBP25 in non-transformed systems. As such, this study aimed to investigate potential roles for FKBP25 in myoblast viability, migration and differentiation and in adaptation of mature skeletal muscle. Doxycycline-inducible FKBP25 knockdown in C2C12 myoblasts revealed an increase in cell accumulation/viability and migration in vitro that was independent of alterations in tubulin dynamics; however, FKBP25 knockdown had no discernible impact on myoblast differentiation into myotubes. Finally, a series of in vivo models of muscle adaptation were assessed, where it was observed that FKBP25 protein expression was increased in hypertrophy and regeneration conditions (chronic mechanical overload and the mdx model of Duchenne muscular dystrophy) but decreased in an atrophy model (denervation). Overall, the findings of this study establish FKBP25 as a regulator of myoblast viability and migration, with possible implications for satellite cell proliferation and migration and muscle regeneration, and as a potential regulator of in vivo skeletal muscle adaptation.

A doxycycline-inducible, tissue-specific aromatase-expressing transgenic mouse.

Aromatase converts androgens to estrogens and it is expressed in gonads and non-reproductive tissues (e.g. brain and adipose tissues). As circulating levels of estrogens in males are low, we hypothesize that local estrogen production is important for the regulation of physiological functions (e.g. metabolism) and pathological development (e.g. breast and prostate cancers) by acting in a paracrine and/or intracrine manner. We generated a tissue-specific doxycycline-inducible, aromatase transgenic mouse to test this hypothesis. The transgene construct (pTetOAROM) consists of a full-length human aromatase cDNA (hAROM) and a luciferase gene under the control of a bi-directional tetracycline-responsive promoter (pTetO), which is regulated by transactivators (rtTA or tTA) and doxycycline. Our in vitro studies using MBA-MB-231tet cells stably expressing rtTA, showed that doxycycline treatment induced transgene expression of hAROM transcripts by 17-fold (P = 0.01), aromatase activity by 26-fold, (P = 0.0008) and luciferase activity by 9.6-fold (P = 0.0006). Pronuclear microinjection of the transgene generated four pTetOAROM founder mice. A male founder was bred with a female mammary gland-specific rtTA mouse (MMTVrtTA) to produce MMTVrtTA-pTetOAROM double-transgenic mice. Upon doxycycline treatment via drinking water, human aromatase expression was detected by RT-PCR, specifically in mammary glands, salivary glands and seminal vesicles of double-stransgenic mice. Luciferase expression and activity was detected in these tissues by in vivo bioluminescence imaging, in vitro luciferase assay and RT-PCR. In summary, we generated a transgenic mouse model that expresses the human aromatase transgene in a temporal- and spatial-specific manner, which will be a useful model to study the physiological importance of local estrogen production.

Enhancing the Bioactivity of Bicyclic Peptides Targeted to Grb7-SH2 by Restoring Cell Permeability.

The development of peptide inhibitors against intracellular targets depends upon the dual challenge of achieving a high affinity and specificity for the target and maintaining cellular permeability for biological activity. Previous efforts to develop bicyclic peptides targeted to the Grb7 signalling protein implicated in HER2+ve cancer progression have resulted in improved affinity. However, these same peptides demonstrated a lowered activity due to their decreased ability to penetrate cell membranes. Here, we report the testing of a new series of bicyclic G7 peptides designed to possess improved bioactivity. We discovered that the incorporation of two amino acids (Phe-Pro, Phe-Trp or Phe-Arg) within the bicyclic peptide framework maintains an enhanced binding affinity for the Grb7-SH2 domain compared to that of the first-generation monocyclic peptide G7-18NATE. Structure determination using X-ray crystallography revealed that the mode of binding by the expanded bicyclic G7 peptide is analogous to that of G7-18NATE. Interestingly, while the bicyclic peptide containing Phe-Trp did not display the highest affinity for Grb7-SH2 in the series, it was the most potent inhibitor of HER2+ve SKBR3 breast cancer cell migration when coupled to Penetratin. Together, this demonstrates that peptide flexibility as well as the amino acid tryptophan can play important roles in the uptake of peptides into the cell.

Regulation of a Novel Splice Variant of Early Growth Response 4 (EGR4-S) by HER+ Signalling and HSF1 in Breast Cancer.

The zinc finger transcription factor EGR4 has previously been identified as having a critical role in the proliferation of small cell lung cancer. Here, we have identified a novel, shortened splice variant of this transcription factor (EGR4-S) that is regulated by Heat Shock Factor-1 (HSF1). Our findings demonstrate that the shortened variant (EGR4-S) is upregulated with high EGFR, HER2, and H-Rasv12-expressing breast cell lines, and its expression is inhibited in response to HER pathway inhibitors. Protein and mRNA analyses of HER2+ human breast tumours indicated the novel EGR4-S splice variant to be preferentially expressed in tumour tissue and not detectable in patient-matched normal tissue. Knockdown of EGR4-S in the HER2-amplified breast cancer cell line SKBR3 reduced cell growth, suggesting that EGR4-S supports the growth of HER2+ tumour cells. In addition to chemical inhibitors of the HER2 pathway, EGR4-S expression was also found to be suppressed by chemical stressors and the overexpression of HSF1. Under these conditions, reduced EGR4-S levels were associated with the observed lower cell growth rate, but the augmentation of properties associated with higher metastatic potential. Taken together, these findings identify EGR4-S as a potential biomarker for HER2 pathway activation in human tumours that is regulated by HSF1.

Influenza A virus facilitates Streptococcus pneumoniae transmission and disease.

Streptococcus pneumoniae (the pneumococcus) kills approximately 1.6 million people annually. Pneumococcal infections predominantly manifest as pneumonia, sepsis, meningitis, and otitis media. S. pneumoniae is also a member of the normal nasopharyngeal flora, colonizing up to 80% of children. Infection with influenza A virus (IAV) has been associated with both pneumococcal disease and transmission. However, to date no animal model has been available to investigate the role of IAV in the spread of S. pneumoniae. Here we investigate pneumococcal-influenza synergism with a particular focus on the role of IAV on pneumococcal transmission. Infant mice were colonized with S. pneumoniae and subsequently infected with IAV 3 d later. Using this novel model we show increased pneumococcal colonization and disease in the presence of IAV. Notably, in vivo imaging showed that IAV was essential for the transmission of S. pneumoniae from colonized ("index") mice to their naive cohoused littermates ("contacts"). Transmission occurred only when all mice were infected with IAV and was prevented when an IAV-neutralizing antibody was used to inhibit IAV replication in either index mice or contact mice. Together, these data provide novel insights into pneumococcal-influenza synergism and may indicate a previously unappreciated role of IAV in the spread of S. pneumoniae.

Benzopyrazine derivatives: A novel class of growth factor receptor bound protein 7 antagonists.

Growth factor receptor bound protein 7 (Grb7) is an adapter protein that functions as a downstream effector of growth factor mediated signal transduction. Over-expression of Grb7 has been implicated in a variety of cancers such as breast, blood, pancreatic, esophageal, and gastric carcinomas. Inhibition of Grb7 has been shown to reduce the migratory and proliferative potential of these cancers, making it an attractive therapeutic target. Starting with a known peptide antagonist, the present work reports the application of a succession of computational ligand design tools comprising a ligand shape based similarity search, molecular docking and a 2D-similarity search to identify small molecular antagonists of the Grb7-SH2 domain from the NCI chemical database. Binding to the Grb7-SH2 domain was then experimentally tested using melting point shift assays and isothermal titration calorimetry. Overall, a total of 11 benzopyrazine based small molecular antagonists were identified with affinity for the Grb7-SH2 domain. Representative compounds tested using ITC were revealed to possess moderate binding affinity in the low micromolar range. Finally, the lead compound (NSC642056) was found to reduce the growth of a Grb7-expressing breast cancer cell line with an IC(50) of 86µM. It is expected that the identified antagonists will be useful additions to further explore the function of Grb7 and for the development of inhibitors with therapeutic potential.

Netrin-1-like-immunoreactivity Coexpresses With DCC and Has a Differential Level in the Myenteric Cholinergic and Nitrergic Neurons of the Adult Mouse Colon.

Netrin-1 is a potent axonal and neuronal guidance cue in the developing nervous system. Netrin-1 functions are mediated by its receptors, such as deleted in colorectal cancer (DCC) present on axons and neurons. Localization of DCC and Netrin-1 on various types of enteric neurons and their role in the mature enteric nervous system is unknown. The results of our study revealed that almost all enteric neurons and processes express DCC and Netrin-1 in the adult mice. Netrin-1-like-immunoreactivity (IR) was detected in the cytoplasm of neurons with some showing strong or weak staining. The majority of Netrin-1-like-immunoreactive enteric neurons were choline acetyltransferase (ChAT)-positive. However, ~19% of neurons were strongly Netrin-1-like-positive but ChAT-negative while ~8% of neurons were Netrin-1-like-negative but strongly ChAT-positive. In contrast, almost all nitric oxide synthase (nNOS)-positive enteric neurons displayed strong Netrin-1-like-IR. This differential intensity of Netrin-1 expression in the myenteric neurons might determine major neuronal subtypes regulating intestinal motility, ChAT-IR excitatory, and nNOS-IR inhibitory muscle motor and interneurons. This is the first study demonstrating the localization of DCC and Netrin-1 in the colonic myenteric plexus of the adult mice and their expression level determining two major neuronal subtypes regulating intestinal motility.

The importance of developing therapies targeting the biological spectrum of metastatic disease.

Great progress has been made in cancer therapeutics. However, metastasis remains the predominant cause of death from cancer. Importantly, metastasis can manifest many years after initial treatment of the primary cancer. This is because cancer cells can remain dormant before forming symptomatic metastasis. An important question is whether metastasis research should focus on the early treatment of metastases, before they are clinically evident ("overt"), or on developing treatments to stop overt metastasis (stage IV cancer). In this commentary we want to clarify why it is important that all avenues of treatment for stage IV patients are developed. Indeed, future treatments are expected to go beyond the mere shrinkage of overt metastases and will include strategies that prevent disseminated tumor cells from emerging from dormancy.

Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective.

Many heat shock proteins (HSPs) are essential to survival as a consequence of their role as molecular chaperones, and play a critical role in maintaining cellular proteostasis by integrating the fundamental processes of protein folding and degradation. HSPs are arguably among the most prominent classes of proteins that have been broadly linked to many human disorders, with changes in their expression profile and/or intracellular/extracellular location now being described as contributing to the pathogenesis of a number of different diseases. Although the concept was initially controversial, it is now widely accepted that HSPs have additional biological functions over and above their role in proteostasis (so-called 'protein moonlighting'). Most importantly, these new insights are enlightening our understanding of biological processes in health and disease, and revealing novel and exciting therapeutic opportunities. This theme issue draws on therapeutic insights from established research on HSPs in cancer and other non-communicable disorders, with an emphasis on how the intracellular function of HSPs contrasts with their extracellular properties and function, and interrogates their potential diagnostic and therapeutic value to the prevention, management and treatment of chronic diseases.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

The type I collagen induction of MT1-MMP-mediated MMP-2 activation is repressed by alphaVbeta3 integrin in human breast cancer cells.

The influence of alphaVbeta3 integrin on MT1-MMP functionality was studied in human breast cancer cells of differing beta3 integrin status. Overexpression of beta3 integrin caused increased cell surface expression of alphaV integrin and increased cellular adhesion to extracellular matrix (ECM) substrates in BT-549, MDA-MB-231 and MCF-7 cells. beta3 integrin expression also enhanced the migration of breast cancer cells on ECM substrates and enhanced collagen gel contraction. In vivo, alphaVbeta3 cooperated with MT1-MMP to increase the growth of MCF-7 cells after orthotopic inoculation in immunocompromised mice, but had no influence on in vitro proliferation. Despite these stimulatory effects, overexpression of beta3 integrin suppressed the type I collagen (Col I) induced MMP-2 activation in all breast cancer cell lines analyzed. This was also evident in extracts from the MCF-7 tumors in vivo, where MMP-2 activation was stimulated by MT1-MMP transfection, but attenuated with beta3 integrin expression. Although our studies confirm important biological effects of alphaVbeta3 integrin on enhancing cell adhesion and migration, ECM remodeling and tumor growth, beta3 integrin caused reduced MMP-2 activation in response to Col I in vitro, which appears to be physiologically relevant, as it was also seen in tumor xenografts in vivo. The reduction of MMP-2 activation (and thus MT1-MMP activity) by alphaVbeta3 in response to Col I may be important in scenarios where cells which are activated for matrix degradation need to preserve some pericellular collagen, perhaps as a substrate for cell adhesion and migration, thus maintaining a balanced level of proteolysis required for efficient tumor growth.

Osteoclast inhibitory lectin (OCIL) inhibits osteoblast differentiation and function in vitro.

Osteoclast inhibitory lectin (OCIL) is a type II C-type lectin and binds NK cell-associated receptor Nkrp1d and sulfated glycosaminoglycans. OCIL is expressed by several cell types found in bone and inhibits osteoclast differentiation. To determine whether OCIL may have wider effects on bone metabolism, we examined the effects of recombinant soluble OCIL on cultured osteoblasts and pre-osteoblastic KUSA O cells. Although OCIL did not affect osteoblast proliferation or apoptosis, or the formation of alkaline phosphatase positive colonies in cultured bone marrow, OCIL profoundly inhibited mineralization by primary osteoblasts and KUSA O cells in vitro. Analysis of ascorbate-treated KUSA O cells showed that addition of OCIL reduced bone sialoprotein (BSP), osterix and osteocalcin mRNA expression, as well as alkaline phosphatase activity while, in contrast, expression of markers associated with the earlier stages of osteoblast maturation or the transcription factors Runx2, ATF4 and c-fos were not affected by OCIL treatment. Indeed, osteocalcin expression was strongly inhibited within 3 days in a dose-dependent manner, although after subsequent removal of OCIL, osteocalcin mRNA levels recovered within 4 days. OCIL treatment also reduced osteocalcin expression in BMP-2 stimulated C2C12 cells. In support of a role for OCIL in mineralization, OCIL anti-sense oligonucleotide treatment of KUSA O cells increased mineralization and osteocalcin expression. In addition, insulin-, dexamethasone- and IBMX-stimulated KUSA O cells undergo adipocyte differentiation and OCIL treatment greatly suppressed this process. Consistent with this, OCIL also reduced adiponectin and resistin mRNA expression in these cells. Our data indicate that OCIL reduces osteoblastic function in vitro and this may be due to an inhibitory effect on osteoblast maturation. In addition, the reduction of adipocyte formation in KUSA O cells by OCIL indicates that OCIL may have wider effects on the mesenchymal lineage that may be important for both bone metabolism and other connective tissue functions.