Probir Chakravarty

Interleukin-6 as a Therapeutic Target in Human Ovarian Cancer

Purpose: We investigated whether inhibition of interleukin 6 (IL-6) has therapeutic activity in ovarian cancer via abrogation of a tumor-promoting cytokine network. Experimental Design: We combined preclinical and in silico experiments with a phase 2 clinical trial of the anti-IL-6 antibody siltuximab in patients with platinum-resistant ovarian cancer. Results: Automated immunohistochemistry on tissue microarrays from 221 ovarian cancer cases showed that intensity of IL-6 staining in malignant cells significantly associated with poor prognosis. Treatment of ovarian cancer cells with siltuximab reduced constitutive cytokine and chemokine production and also inhibited IL-6 signaling, tumor growth, the tumor-associated macrophage infiltrate and angiogenesis in IL-6–producing intraperitoneal ovarian cancer xenografts. In the clinical trial, the primary endpoint was response rate as assessed by combined RECIST and CA125 criteria. One patient of eighteen evaluable had a partial response, while seven others had periods of disease stabilization. In patients treated for 6 months, there was a significant decline in plasma levels of IL-6–regulated CCL2, CXCL12, and VEGF. Gene expression levels of factors that were reduced by siltuximab treatment in the patients significantly correlated with high IL-6 pathway gene expression and macrophage markers in microarray analyses of ovarian cancer biopsies. Conclusion: IL-6 stimulates inflammatory cytokine production, tumor angiogenesis, and the tumor macrophage infiltrate in ovarian cancer and these actions can be inhibited by a neutralizing anti-IL-6 antibody in preclinical and clinical studies. Clin Cancer Res; 17(18); 6083–96. ©2011 AACR.

The tumor-promoting actions of TNF-α involve TNFR1 and IL-17 in ovarian cancer in mice and humans

Cytokines orchestrate the tumor-promoting interplay between malignant cells and the immune system. In many experimental and human cancers, the cytokine TNF-alpha is an important component of this interplay, but its effects are pleiotropic and therefore remain to be completely defined. Using a mouse model of ovarian cancer in which either TNF receptor 1 (TNFR1) signaling was manipulated in different leukocyte populations or TNF-alpha was neutralized by antibody treatment, we found that this inflammatory cytokine maintained TNFR1-dependent IL-17 production by CD4+ cells and that this led to myeloid cell recruitment into the tumor microenvironment and enhanced tumor growth. Consistent with this, in patients with advanced cancer, treatment with the TNF-alpha-specific antibody infliximab substantially reduced plasma IL-17 levels. Furthermore, expression of IL-1R and IL-23R was downregulated in CD4+CD25- cells isolated from ascites of ovarian cancer patients treated with infliximab. We have also shown that genes ascribed to the Th17 pathway map closely with the TNF-alpha signaling pathway in ovarian cancer biopsy samples, showing particularly high levels of expression of genes encoding IL-23, components of the NF-kappaB system, TGF-beta1, and proteins involved in neutrophil activation. We conclude that chronic production of TNF-alpha in the tumor microenvironment increases myeloid cell recruitment in an IL-17-dependent manner that contributes to the tumor-promoting action of this proinflammatory cytokine.

Cyclooxygenase-Dependent Tumor Growth through Evasion of Immunity

Summary The mechanisms by which melanoma and other cancer cells evade anti-tumor immunity remain incompletely understood. Here, we show that the growth of tumors formed by mutant BrafV600E mouse melanoma cells in an immunocompetent host requires their production of prostaglandin E2, which suppresses immunity and fuels tumor-promoting inflammation. Genetic ablation of cyclooxygenases (COX) or prostaglandin E synthases in BrafV600E mouse melanoma cells, as well as in NrasG12D melanoma or in breast or colorectal cancer cells, renders them susceptible to immune control and provokes a shift in the tumor inflammatory profile toward classic anti-cancer immune pathways. This mouse COX-dependent inflammatory signature is remarkably conserved in human cutaneous melanoma biopsies, arguing for COX activity as a driver of immune suppression across species. Pre-clinical data demonstrate that inhibition of COX synergizes with anti-PD-1 blockade in inducing eradication of tumors, implying that COX inhibitors could be useful adjuvants for immune-based therapies in cancer patients.

IL6-STAT3-HIF Signaling and Therapeutic Response to the Angiogenesis Inhibitor Sunitinib in Ovarian Clear Cell Cancer

Purpose: Ovarian clear cell adenocarcinoma (OCCA) is an uncommon histotype that is generally refractory to platinum-based chemotherapy. We analyze here the most comprehensive gene expression and copy number data sets, to date, to identify potential therapeutic targets of OCCA. Experimental Design: Gene expression and DNA copy number were carried out using primary human OCCA tumor samples, and findings were confirmed by immunohistochemistry on tissue microarrays. Circulating interleukin (IL) 6 levels were measured in serum from patients with OCCA or high-grade serous cancers and related to progression-free and overall survival. Two patients were treated with sunitinib, and their therapeutic responses were measured clinically and by positron emission tomography. Results: We find specific overexpression of the IL6-STAT3-HIF (interleukin 6-signal transducer and activator of transcription 3-hypoxia induced factor) pathway in OCCA tumors compared with high-grade serous cancers. Expression of PTHLH and high levels of circulating IL6 in OCCA patients may explain the frequent occurrence of hypercalcemia of malignancy and thromboembolic events in OCCA. We describe amplification of several receptor tyrosine kinases, most notably MET, suggesting other potential therapeutic targets. We report sustained clinical and functional imaging responses in two OCCA patients with chemotherapy-resistant disease who were treated with sunitinib, thus showing significant parallels with renal clear cell cancer. Conclusions: Our findings highlight important therapeutic targets in OCCA, suggest that more extensive clinical trials with sunitinib in OCCA are warranted, and provide significant impetus to the growing realization that OCCA is molecularly and clinically distinct to other forms of ovarian cancer. Clin Cancer Res; 17(8); 2538–48. ©2011 AACR.

CLEC-2 signaling via Syk in myeloid cells can regulate inflammatory responses.

Myeloid cells express a plethora of C‐type lectin receptors (CLRs) that can regulate immune responses. CLEC‐2 belongs to the Dectin‐1 sub‐family of CLRs that possess an extracellular C‐type lectin‐like domain and a single intracellular hemITAM motif. CLEC‐2 is highly expressed on mouse and human platelets where it signals via Syk to promote aggregation. We generated a monoclonal antibody (mAb) against mouse CLEC‐2 and found that CLEC‐2 is additionally widely expressed on leukocytes and that its expression is upregulated during inflammation. MAb‐mediated crosslinking of CLEC‐2 leads to hemITAM‐dependent signaling via Syk, Ca2+ and NFAT and, in myeloid cells, modulates the effect of toll‐like receptor (TLR) agonists to selectively potentiate production of IL‐10. A macrophage/dendritic cell‐dependent increase in IL‐10 is also observed in mice given anti‐CLEC‐2 mAb together with LPS. Collectively, these data indicate that CLEC‐2 is expressed in myeloid cells and acts as a Syk‐coupled CLR able to modulate TLR signaling and inflammatory responses.

An antagonist of the chemokine receptor CXCR4 induces mitotic catastrophe in ovarian cancer cells

The chemokine receptor CXCR4 is expressed by malignant cells in ovarian cancer and is implicated in their growth and spread. We report here a unique mechanism of action of a small peptide antagonist of CXCR4 on ovarian cancer cells: induction of cell death by mitotic catastrophe. CTCE-9908 inhibited ovarian cancer cell migration to CXCL12, but on longer incubation, caused cell death in CXCR4-positive cells. CTCE-9908 did not cause apoptosis or cellular senescence, but induced multinucleation, G2-M arrest, and abnormal mitosis in ovarian cancer cells. This suggests that cell death was caused by mitotic catastrophe. Using microarray and Western blot analysis, we showed that CTCE-9908 deregulated DNA damage checkpoint proteins and spindle assembly checkpoint proteins at G2-M phases of the cell cycle. Combination treatment of CTCE-9908 and the drug paclitaxel led to an additive cytotoxicity that also involved mitotic catastrophe. We conclude that CTCE-9908 has a unique mechanism of action in ovarian cancer cells that seems to be CXCR4 specific. [Mol Cancer Ther 2009;8(7):1893–905]

Two mutations in the KINDLIN3 gene of a new leukocyte adhesion deficiency-III patient reveal distinct effects on leukocyte function in vitro

In the disorder leukocyte adhesion deficiency III (LAD-III), integrins on platelets and leukocytes are expressed but fail to function and this leads to severe bleeding and infections at an early age. Mutation in the KINDLIN3 (FERMT3) gene is the cause of LAD-III in patients from the Middle East, Malta, and Turkey. We describe 2 novel homozygous mutations in the KINDLIN3 gene of a new African-American patient that destabilize KINDLIN3 mRNA leading to loss of kindlin-3 protein. Transfection of wild-type (WT) KINDLIN3 cDNA restored integrin-related adhesion and migration in the LAD-III patients T and B lymphocytes. We analyzed the individual mutations separately in vitro to learn more about the function of the kindlin-3 protein. The first G>A mutation gives rise to a Gly308Arg change at the end of FERM (protein 4.1, ezrin, radixin, moesin) subdomain 2, and the second mutation is a base deletion causing early termination within the pleckstrin homology (PH) domain. This second mutation prevented membrane association of kindlin-3 and did not restore either adhesion or migration, whereas the FERM subdomain 2 mutation affected only migration. Thus, these LAD-III patient mutations have highlighted functionally important regions of kindlin-3 that alter leukocyte integrin-dependent function in 2 distinct ways.

Identification of an LGP2-associated MDA5 agonist in picornavirus-infected cells

The RIG-I-like receptors RIG-I, LGP2, and MDA5 initiate an antiviral response that includes production of type I interferons (IFNs). The nature of the RNAs that trigger MDA5 activation in infected cells remains unclear. Here, we purify and characterise LGP2/RNA complexes from cells infected with encephalomyocarditis virus (EMCV), a picornavirus detected by MDA5 and LGP2 but not RIG-I. We show that those complexes contain RNA that is highly enriched for MDA5-stimulatory activity and for a specific sequence corresponding to the L region of the EMCV antisense RNA. Synthesis of this sequence by in vitro transcription is sufficient to generate an MDA5 stimulatory RNA. Conversely, genomic deletion of the L region in EMCV generates viruses that are less potent at stimulating MDA5-dependent IFN production. Thus, the L region antisense RNA of EMCV is a key determinant of innate immunity to the virus and represents an RNA that activates MDA5 in virally-infected cells. DOI: http://dx.doi.org/10.7554/eLife.01535.001

Activation of ULK Kinase and Autophagy by GABARAP Trafficking from the Centrosome Is Regulated by WAC and GM130

Summary Starvation-induced autophagy requires activation of the ULK complex at the phagophore. Two Golgi proteins, WAC and GM130, regulate autophagy, however their mechanism of regulation is unknown. In search of novel interaction partners of WAC, we found that GM130 directly interacts with WAC, and this interaction is required for autophagy. WAC is bound to the Golgi by GM130. WAC and GM130 interact with the Atg8 homolog GABARAP and regulate its subcellular localization. GABARAP is on the pericentriolar matrix, and this dynamic pool contributes to autophagosome formation. Tethering of GABARAP to the Golgi by GM130 inhibits autophagy, demonstrating an unexpected role for a golgin. WAC suppresses GM130 binding to GABARAP, regulating starvation-induced centrosomal GABARAP delivery to the phagophore. GABARAP, unlipidated and lipidated, but not LC3B, GABARAPL1, and GATE-16, specifically promotes ULK kinase activation dependent on the ULK1 LIR motif, elucidating a unique non-hierarchical role for GABARAP in starvation-induced activation of autophagy.

Dysregulation of gene expression as a cause of Cockayne syndrome neurological disease

Significance Cockayne syndrome (CS) is an autosomal-recessive, multisystem disorder characterized by neurological disease, growth failure, developmental abnormalities, photosensitivity, and degeneration of organ systems such as the ear and eye, including cataracts. Most patients with CS carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we provide evidence from human and mouse cell models, as well as brain tissue from patients with CS, that the involvement of CSB in regulating gene expression can explain several features of CS. Together, our data suggest that dysregulation of gene regulatory networks rather than DNA repair defects may be the main cause of neurological symptoms in CS. Cockayne syndrome (CS) is a multisystem disorder with severe neurological symptoms. The majority of CS patients carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled nucleotide excision repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we investigate the connection between the neuropathology of CS and dysregulation of gene expression. Transcriptome analysis of human fibroblasts revealed that even in the absence of DNA damage, CSB affects the expression of thousands of genes, many of which are neuronal genes. CSB is present in a significant subset of these genes, suggesting that regulation is direct, at the level of transcription. Importantly, reprogramming of CS fibroblasts to neuron-like cells is defective unless an exogenous CSB gene is introduced. Moreover, neuroblastoma cells from which CSB is depleted show defects in gene expression programs required for neuronal differentiation, and fail to differentiate and extend neurites. Likewise, neuron-like cells cannot be maintained without CSB. Finally, a number of disease symptoms may be explained by marked gene expression changes in the brain of patients with CS. Together, these data point to dysregulation of gene regulatory networks as a cause of the neurological symptoms in CS.