USP7 inhibitors suppress tumour neoangiogenesis and promote synergy with immune checkpoint inhibitors by downregulating fibroblast VEGF.

BACKGROUND: Understanding how to modulate the microenvironment of tumors that are resistant to immune checkpoint inhibitors represents a major challenge in oncology.Here we investigate the ability of USP7 inhibitors to reprogram the tumor microenvironment (TME) by inhibiting secretion of vascular endothelial growth factor (VEGF) from fibroblasts. METHODS: To understand the role played by USP7 in the TME, we systematically evaluated the effects of potent, selective USP7 inhibitors on co-cultures comprising components of the TME, using human primary cells. We also evaluated the effects of USP7 inhibition on tumor growth inhibition in syngeneic models when dosed in combination with immune checkpoint inhibitors (ICIs). RESULTS: Abrogation of VEGF secretion from fibroblasts in response to USP7 inhibition resulted in inhibition of tumor neoangiogenesis and increased tumor recruitment of CD8-positive T-lymphocytes, leading to significantly improved sensitivity to immune checkpoint inhibitors. In syngeneic models, treatment with USP7 inhibitors led to striking tumor responses resulting in significantly improved survival. CONCLUSIONS: USP7-mediated reprograming of the TME is not linked to its previously characterized role in modulating MDM2 but does require p53 and UHRF1 in addition to the well-characterized VEGF transcription factor, HIF-1α. This represents a function of USP7 that is unique to fibroblasts, and which is not observed in cancer cells or other components of the TME. Given the potential for USP7 inhibitors to transform "immune desert" tumors into "immune responsive" tumors, this paves the way for a novel therapeutic strategy combining USP7 inhibitors with immune checkpoint inhibitors (ICIs).

Calreticulin exploits TGF-ß for extracellular matrix induction engineering a tissue regenerative process.

Topical application of extracellular calreticulin (eCRT), an ER chaperone protein, in animal models enhances wound healing and induces tissue regeneration evidenced by epidermal appendage neogenesis and lack of scarring. In addition to chemoattraction of cells critical to the wound healing process, eCRT induces abundant neo-dermal extracellular matrix (ECM) formation by 3 days post-wounding. The purpose of this study was to determine the mechanisms involved in eCRT induction of ECM. In vitro, eCRT strongly induces collagen I, fibronectin, elastin, α-smooth muscle actin in human adult dermal (HDFs) and neonatal fibroblasts (HFFs) mainly via TGF-ß canonical signaling and Smad2/3 activation; RAP, an inhibitor of LRP1 blocked eCRT ECM induction. Conversely, eCRT induction of α5 and ß1 integrins was not mediated by TGF-ß signaling nor inhibited by RAP. Whereas eCRT strongly induces ECM and integrin α5 proteins in K41 wild-type mouse embryo fibroblasts (MEFs), CRT null MEFs were unresponsive. The data show that eCRT induces the synthesis and release of TGF-ß3 first via LRP1 or other receptor signaling and later induces ECM proteins via LRP1 signaling subsequently initiating TGF-ß receptor signaling for intracellular CRT (iCRT)-dependent induction of TGF-ß1 and ECM proteins. In addition, TGF-ß1 induces 2-3-fold higher level of ECM proteins than eCRT. Whereas eCRT and iCRT converge for ECM induction, we propose that eCRT attenuates TGF-ß-mediated fibrosis/scarring to achieve tissue regeneration.

The crosstalk between breast carcinoma-associated fibroblasts and cancer cells promotes RhoA-dependent invasion via IGF-1 and PAI-1.

Carcinoma-associated fibroblasts (CAFs) can remodel the extracellular matrix to promote cancer cell invasion, but the paracrine signaling between CAFs and cancer cells that regulates tumor cell migration remains to be identified. To determine how the interaction between CAFs and cancer cells modulates the invasiveness of cancer cells, we developed a 3-dimensional co-culture model composed of breast cancer (BC) MDA-MB-231 cell spheroids embedded in a collagen gel with and without CAFs. We found that the crosstalk between CAFs and cancer cells promotes invasion by stimulating the scattering of MDA-MB-231 cells, which was dependent on RhoA/ROCK/phospho MLC signaling in cancer cells but independent of RhoA in CAFs. The activation of RhoA/ROCK in cancer cells activates MLC and increases migration, while the genetic-down-regulation of RhoA and pharmacological inhibition of ROCK reduced cell scattering and invasion. Two distinct mechanisms induced the activation of the RhoA/ROCK pathway in MDA-MB-231 cells, the secretion of IGF-1 by CAFs and the upregulation of PAI-1 in cancer cells. In an orthotopic model of BC, IGF-1R inhibition decreased the incidence of lung metastasis, while Y27632-inhibition of ROCK enhanced the lung metastasis burden, which was associated with an increased recruitment of CAFs and expression of PAI-1. Thus the crosstalk between CAFs and BC cells increases the secretion of IGF-1 in CAFs and PAI-1 activity in cancer cells. Both IGF1 and PAI-1 activate RhoA/ROCK signaling in cancer cells, which increases cell scattering and invasion.

Hormonal and Growth Regulation of Epithelial and Stromal Cells From the Normal and Malignant Endometrium by Pigment Epithelium-Derived Factor.

We discovered that pigment epithelium-derived factor (PEDF)-null mice have endometrial hyperplasia, the precursor to human type I endometrial cancer (ECA), which is etiologically linked to unopposed estrogen (E2), suggesting that this potent antiangiogenic factor might contribute to dysregulated growth and the development of type I ECA. Treatment of both ECA cell lines and primary ECA cells with recombinant PEDF dose dependently decreased cellular proliferation via an autocrine mechanism by blocking cells in G1 and G2 phases of the cell cycle. Consistent with the known opposing effects of E2 and progesterone (Pg) on endometrial proliferation, Pg increases PEDF protein synthesis and release, whereas E2 has the converse effect. Using PEDF luciferase promoter constructs containing two Pg and one E2 response elements, E2 reduced and Pg increased promoter activity due to distal response elements. Furthermore, E2 decreases and Pg increases PEDF secretion into conditioned media (CM) by both normal endometrial stromal fibroblasts (ESFs) and cancer-associated fibroblasts (CAFs), but only CM from ESFs mediated growth-inhibitory activity of primary endometrial epithelial cells (EECs). In addition, in cocultures with primary EECs, Pg-induced growth inhibition is mediated by ESFs, but not CAFs. This is consistent with reduced levels of Pg receptors on CAFs surrounding human malignant glands in vivo. Taken together, the data suggest that PEDF is a hormone-regulated negative autocrine mediator of endometrial proliferation, and that paracrine growth inhibition by soluble factors, possibly PEDF, released by ESFs in response to Pg, but not CAFs, exemplifies a tumor microenvironment that contributes to the pathogenesis of ECA.

Anti-VEGF therapy induces ECM remodeling and mechanical barriers to therapy in colorectal cancer liver metastases.

The survival benefit of anti-vascular endothelial growth factor (VEGF) therapy in metastatic colorectal cancer (mCRC) patients is limited to a few months because of acquired resistance. We show that anti-VEGF therapy induced remodeling of the extracellular matrix with subsequent alteration of the physical properties of colorectal liver metastases. Preoperative treatment with bevacizumab in patients with colorectal liver metastases increased hyaluronic acid (HA) deposition within the tumors. Moreover, in two syngeneic mouse models of CRC metastasis in the liver, we show that anti-VEGF therapy markedly increased the expression of HA and sulfated glycosaminoglycans (sGAGs), without significantly changing collagen deposition. The density of these matrix components correlated with increased tumor stiffness after anti-VEGF therapy. Treatment-induced tumor hypoxia appeared to be the driving force for the remodeling of the extracellular matrix. In preclinical models, we show that enzymatic depletion of HA partially rescued the compromised perfusion in liver mCRCs after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and chemotherapy. These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.

TGF-ß activates APC through Cdh1 binding for Cks1 and Skp2 proteasomal destruction stabilizing p27kip1 for normal endometrial growth.

We previously reported that aberrant TGF-ß/Smad2/3 signaling in endometrial cancer (ECA) leads to continuous ubiquitylation of p27(kip1)(p27) by the E3 ligase SCF-Skp2/Cks1 causing its degradation, as a putative mechanism involved in the pathogenesis of this cancer. In contrast, normal intact TGF-ß signaling prevents degradation of nuclear p27 by SCF-Skp2/Cks1 thereby accumulating p27 to block Cdk2 for growth arrest. Here we show that in ECA cell lines and normal primary endometrial epithelial cells, TGF-ß increases Cdh1 and its binding to APC/C to form the E3 ligase complex that ubiquitylates Cks1 and Skp2 prompting their proteasomal degradation and thus, leaving p27 intact. Knocking-down Cdh1 in ECA cell lines increased Skp2/Cks1 E3 ligase activity, completely diminished nuclear and cytoplasmic p27, and obviated TGF-ß-mediated inhibition of proliferation. Protein synthesis was not required for TGF-ß-induced increase in nuclear p27 and decrease in Cks1 and Skp2. Moreover, half-lives of Cks1 and Skp2 were extended in the Cdh1-depleted cells. These results suggest that the levels of p27, Skp2 and Cks1 are strongly or solely regulated by proteasomal degradation. Finally, an inverse relationship of low p27 and high Cks1 in the nucleus was shown in patients in normal proliferative endometrium and grade I-III ECAs whereas differentiated secretory endometrium showed the reverse. These studies implicate Cdh1 as the master regulator of TGF-ß-induced preservation of p27 tumor suppressor activity. Thus, Cdh1 is a potential therapeutic target for ECA and other human cancers showing an inverse relationship between Cks1/Skp2 and p27 and/or dysregulated TGF-ß signaling.

High-level secretion of native recombinant human calreticulin in yeast.

BACKGROUND: Calreticulin (CRT) resides in the endoplasmic reticulum (ER) and functions to chaperone proteins, ensuring proper folding, and intracellular Ca(2+) homeostasis. Emerging evidence shows that CRT is a multifunctional protein with significant roles in physiological and pathological processes with presence both inside and outside of the ER, including the cell surface and extracellular space. These recent findings suggest the possible use of this ER chaperone in development of new therapeutic pharmaceuticals. Our study was focused on human CRT production in two yeast species, Saccharomyces cerevisiae and Pichia pastoris. RESULTS: Expression of a full-length human CRT precursor including its native signal sequence resulted in high-level secretion of mature recombinant protein into the culture medium by both S. cerevisiae and P. pastoris. To ensure the structural and functional quality of the yeast-derived CRTs, we compared yeast-secreted human recombinant CRT with native CRT isolated from human placenta. In ESI-MS (electrospray ionization mass spectrometry), both native and recombinant full-length CRT showed an identical molecular weight (mass) of 46,466 Da and were monomeric by non-denaturing PAGE. Moreover, limited trypsin digestion yielded identical fragment patterns of calcium-binding recombinant and native CRT suggesting that the yeast-derived CRT was correctly folded. Furthermore, both native and recombinant CRT induced cellular proliferation (MTS assay) and migration of human dermal fibroblasts (in vitro wound healing assay) with the same specific activities (peak responses at 1-10 ng/ml) indicating that the functional integrity of yeast-derived CRT was completely preserved. Simple one-step purification of CRT from shake-flask cultures resulted in highly pure recombinant CRT protein with yields reaching 75 % of total secreted protein and with production levels of 60 and 200 mg/l from S. cerevisiae and P. pastoris, respectively. Finally, cultivation of P. pastoris in a bioreactor yielded CRT secretion titer to exceed 1.5 g/l of culture medium. CONCLUSIONS: Yeasts are able to correctly process and secrete large amounts of mature recombinant human CRT equally and fully biologically active as native human CRT. This allows efficient production of high-quality CRT protein in grams per liter scale.

RhoA mediates flow-induced endothelial sprouting in a 3-D tissue analogue of angiogenesis.

Endothelial cells (ECs) integrate signals from the local microenvironment to guide their behaviour. RhoA is involved in vascular endothelial growth factor (VEGF)-driven angiogenesis, but its role in mechanotransduction during sprouting has not been established. Using dominant negative cell transfections in a microfluidic device that recapitulates angiogenic sprouting, we show that endothelial cells respond to interstitial flow in a RhoA-dependent manner while invading a 3-D extracellular matrix. Furthermore, RhoA regulates flow-induced, but not VEGF gradient-induced, tip cell filopodial extensions. Thus, RhoA pathways mediate mechanically-activated but not VEGF-induced endothelial morphogenesis.

Molecular changes in mesothelioma with an impact on prognosis and treatment.

CONTEXT: In recent decades, research on malignant pleural mesothelioma (MPM) has been developed to improve patients' outcomes by increasing the level of confidence in MPM diagnosis and prognosis. OBJECTIVE: To summarize data on genetic and epigenetic abnormalities in MPM that may be of interest for a better management of patients with MPM. DATA SOURCES: Data were obtained from scientific publications on genetic and epigenetic abnormalities in MPM by studying gene mutations, DNA methylation, and gene and microRNA expression profiling. CONCLUSIONS: Molecular changes in MPM consist in altered expression and in activation or inactivation of critical genes in oncogenesis, especially tumor suppressor genes at the INK4 and NF2 loci. Activation of membrane receptor tyrosine kinases and deregulation of signaling pathways related to differentiation, survival, proliferation, apoptosis, cell cycle control, metabolism, migration, and invasion have been demonstrated. Alterations that could be targeted at a global level (methylation) have been recently reported. Experimental research has succeeded especially in abolishing proliferation and triggering apoptosis in MPM cells. So far, targeted clinical approaches focusing on receptor tyrosine kinases have had limited success. Molecular analyses of series of MPM cases have shown that defined alterations are present in MPM subsets, consistent with interindividual variations of molecular alterations, and suggesting that identification of patient subgroups will be essential to develop more specific therapies.

Mesothelioma: Do asbestos and carbon nanotubes pose the same health risk?

Carbon nanotubes (CNTs), the product of new technology, may be used in a wide range of applications. Because they present similarities to asbestos fibres in terms of their shape and size, it is legitimate to raise the question of their safety for human health. Recent animal and cellular studies suggest that CNTs elicit tissue and cell responses similar to those observed with asbestos fibres, which increases concern about the adverse biological effects of CNTs. While asbestos fibres' mechanisms of action are not fully understood, sufficient results are available to develop hypotheses about the significant factors underlying their damaging effects. This review will summarize the current state of knowledge about the biological effects of CNTs and will discuss to what extent they present similarities to those of asbestos fibres. Finally, the characteristics of asbestos known to be associated with toxicity will be analyzed to address the possible impact of CNTs.

Clinico-pathological features and somatic gene alterations in refractory ceramic fibre-induced murine mesothelioma reveal mineral fibre-induced mesothelioma identities.

Although human malignant mesothelioma (HMM) is mainly caused by asbestos exposure, refractory ceramic fibres (RCFs) have been classified as possibly carcinogenic to humans on the basis of their biological effects in rodents' lung and pleura and in cultured cells. Hence, further investigations are needed to clarify the mechanism of fibre-induced carcinogenicity and to prevent use of harmful particles. In a previous study, mesotheliomas were found in hemizygous Nf2 (Nf2(+/-)) mice exposed to asbestos fibres, and showed similar alterations in genes at the Ink4 locus and in Trp53 as described in HMM. Here we found that Nf2(+/-) mice developed mesotheliomas after intra-peritoneal inoculation of a RCF sample (RCF1). Clinical features in exposed mice were similar to those observed in HMM, showing association between ascite and mesothelioma. Early passages of 12 mesothelioma cell cultures from ascites developed in RCF1-exposed Nf2(+/-) mice demonstrated frequent inactivation by deletion of genes at the Ink4 locus, and low rate of Trp53 point and insertion mutations. Nf2 gene was inactivated in all cultures. In most cases, co-inactivation of genes at the Ink4 locus and Nf2 was found and, at a lower rate, of Trp53 and Nf2. These results are the first to identify mutations in RCF-induced mesothelioma. They suggest that nf2 mutation is complementary of p15(Ink4b), p16(Ink4a) and p19(Arf) or p53 mutations and show similar profile of gene alterations resulting from exposure to ceramic or asbestos fibres in Nf2(+/-) mice, also consistent with the one found in HMM. These somatic genetic changes define different pathways of mesothelial cell transformation.