No abstract

Increasing chemotherapy delivery to tumors, while enhancing drug uptake and reducing side effects, is a primary goal of cancer research. In mouse and human cancer models in vivo, we show that coadministration of low-dose Cilengitide and Verapamil increases tumor angiogenesis, leakiness, blood flow, and Gemcitabine delivery. This approach reduces tumor growth, metastasis, and minimizes side effects while extending survival. At a molecular level, this strategy alters Gemcitabine transporter and metabolizing enzyme expression levels, enhancing the potency of Gemcitabine within tumor cells in vivo and in vitro. Thus, the dual action of low-dose Cilengitide, in vessels and tumor cells, improves chemotherapy efficacy. Overall, our data demonstrate that vascular promotion therapy is a means to improve cancer treatment.

Chemoresistance is a serious limitation of cancer treatment1. Until recently, almost all the work done to study this limitation has been restricted to tumour cells2. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically.

The involvement of Toll-like receptor 4 (TLR4) in immunity against human herpesviruses has not been previously demonstrated. We show that infection of endothelial cells with Kaposi sarcoma herpesvirus (KSHV), a human oncogenic virus, leads to rapid suppression of TLR4 expression. This is a mechanism of immune escape as TLR4 mediates innate immunity against KSHV. In vitro, cells lacking TLR4 are more susceptible to KSHV infection, whereas activation of TLR4 protects cells from infection. In vivo, HIV-1-infected individuals carrying a mutant TLR4 allele appear more likely to have multicentric Castleman's disease, a lymphoproliferation associated with enhanced KSHV replication. ERK activation by KSHV structural proteins and the KSHV-encoded vGPCR plays a key role in the TLR4 downregulation, whereas the KSHV vIRF1 also contributes to this effect. Our findings reveal a role for TLR4 in innate immunity against herpesviruses and suggest the potential use of TLR4 agonists for the treatment of KSHV-related neoplasms.

Several studies have explored the potential of targeting tumor angiogenesis in cancer treatment. Anti-angiogenesis monotherapy, which reduces blood vessel numbers, may still hold some promise in cancer treatment, but thus far it has only provided a modest effect on overall survival benefits. When combined with standard chemotherapies, some significant improvements in cancer therapy have been reported. However, anti-angiogenesis therapies can have undesirable effects, including the induction of tumor hypoxia and reduction of delivery of chemotherapeutic drugs. Interestingly, anti-angiogenic drugs, such as bevacizumab, when used at lower doses, can actually induce vascular normalization (that is, they improve blood vessel function and flow) and potentially enhance co-administrated chemotherapeutic drug delivery. Unfortunately, vascular normalization is a difficult approach to apply in clinical settings. Thus, there is an urgent need to explore new approaches for modulating the tumor vasculature. Here, we explore how vascular promotion strategies (which enhance blood vessel numbers and leakiness) may be optimized for combination therapies as an alternative option for cancer treatment.

The TFAP2C transcription factor has been shown to downregulate transcription of the universal cell cycle inhibitor p21 cip (CDKN1A). In examining the mechanism of TFAP2C-mediated repression, we have identified a ternary complex at the proximal promoter containing TFAP2C, the oncoprotein Myc, and the trimethylated lysine 4 of histone H3 (H3K4me3) demethylase, KDM5B. We demonstrated that while TFAP2C and Myc can downregulate the CDKN1A promoter independently, KDM5B acts as a corepressor dependent on the other two proteins. All three factors collaborate for optimal CDKN1A repression, which requires the AP-2 binding site at ؊111/؊103 and KDM5B demethylase activity. Silencing of TFAP2C-KDM5B-Myc led to increased H3K4me3 at the endogenous promoter and full induction of CDKN1A expression. Coimmunoprecipitation assays showed that TFAP2C and Myc associate with distinct domains of KDM5B and the TFAP2C C-terminal 270 amino acids (aa) are required for Myc and KDM5B interaction. Overexpression of all three proteins resulted in forced S-phase entry and attenuation of checkpoint activation, even in the presence of chemotherapy drugs. Since each protein has been linked to poor prognosis in breast cancer, our findings suggest that the TFAP2C-Myc-KDM5B complex promotes cell cycle progression via direct CDKN1A repression, thereby contributing to tumorigenesis and therapy failure.

Defective pericyte-endothelial cell interaction in tumors leads to a chaotic, poorly organized and dysfunctional vasculature. However, the underlying mechanism behind this is poorly studied. Herein, we develop a method that combines magnetic beads and flow cytometry cell sorting to isolate pericytes from tumors and normal adjacent tissues from patients with non-small cell lung cancer (NSCLC) and hepatocellular carcinoma (HCC). Pericytes from tumors show defective blood vessel supporting functions when comparing to those obtained from normal tissues. Mechanistically, combined proteomics and metabolic flux analysis reveals elevated hexokinase 2(HK2)-driven glycolysis in tumor pericytes, which up-regulates their ROCK2-MLC2 mediated contractility leading to impaired blood vessel supporting function. Clinically, high percentage of HK2 positive pericytes in blood vessels correlates with poor patient overall survival in NSCLC and HCC. Administration of a HK2 inhibitor induces pericyte-MLC2 driven tumor vasculature remodeling leading to enhanced drug delivery and efficacy against tumor growth. Overall, these data suggest that glycolysis in tumor pericytes regulates their blood vessel supporting role.

SummaryAlthough the retinoblastoma protein (Rb) functions as a checkpoint in the cell cycle, it also regulates differentiation. It has recently been shown that Rb is acetylated during differentiation; however, the role of this modification has not been identified. Depletion of Rb levels with short hairpin RNA resulted in inhibition of human keratinocyte differentiation, delayed cell cycle exit and allowed cell cycle re-entry. Restoration of Rb levels rescued defects in differentiation and cell cycle exit and re-entry; however, re-expression of Rb with the major acetylation sites mutated did not. During keratinocyte differentiation, acetylation of Rb is mediated by PCAF and it is further shown that PCAF acetyltransferase activity is also required for normal differentiation. The major acetylation sites in Rb are located within the nuclear localization sequence and, although mutation did not alter Rb localization in cycling cells, the mutant is mislocalized to the cytoplasm during differentiation. Studies indicate that acetylation is a mechanism for controlling Rb localization in human keratinocytes, with either reduction of the PCAF or exogenous expression of the deacetylase SIRT1, resulting in mislocalization of Rb. These findings identify PCAF-mediated acetylation of Rb as an event required to retain Rb within the nucleus during keratinocyte differentiation.