Lin Jia

The CXCL12-CXCR4 Chemokine Pathway: A Novel Axis Regulates Lymphangiogenesis

Purpose: Lymphangiogenesis, the growth of lymphatic vessels, contributes to lymphatic metastasis. However, the precise mechanism underlying lymphangiogenesis remains poorly understood. This study aimed to examine chemokine/chemokine receptors that directly contribute to chemoattraction of activated lymphatic endothelial cells (LEC) and tumor lymphangiogenesis. Experimental Design: We used quantitative RT-PCR to analyze specifically expressed chemokine receptors in activated LECs upon stimulation of vascular endothelial growth factor-C (VEGF-C). Subsequently, we established in vitro and in vivo models to show lymphangiogenic functions of the chemokine axis. Effects of targeting the chemokine axis on tumor lymphangiogenesis and lymphatic metastasis were determined in an orthotopic breast cancer model. Results: VEGF-C specifically upregulates CXCR4 expression on lymphangiogenic endothelial cells. Moreover, hypoxia-inducible factor-1α (HIF-1α) mediates the CXCR4 expression induced by VEGF-C. Subsequent analyses identify the ligand CXCL12 as a chemoattractant for LECs. CXCL12 induces migration, tubule formation of LECs in vitro, and lymphangiogenesis in vivo. CXCL12 also stimulates the phosphorylation of intracellular signaling Akt and Erk, and their specific antagonists impede CXCL12-induced chemotaxis. In addition, its level is correlated with lymphatic vessel density in multiple cancer tissues microarray. Furthermore, the CXCL12–CXCR4 axis is independent of the VEGFR-3 pathway in promoting lymphangiogenesis. Intriguingly, combined treatment with anti-CXCL12 and anti-VEGF-C antibodies results in additive inhibiting effects on tumor lymphangiogenesis and lymphatic metastasis. Conclusions: These results show the role of the CXCL12–CXCR4 axis as a novel chemoattractant for LECs in promoting lymphangiogenesis, and support the potential application of combined targeting of both chemokines and lymphangiogenic factors in inhibiting lymphatic metastasis. Clin Cancer Res; 18(19); 5387–98. ©2012 AACR.

MicroRNA-542-3p inhibits tumour angiogenesis by targeting Angiopoietin-2

Angiopoietin‐2 (Angpt2) plays a critical role in angiogenesis and tumour progression. Therapeutic targeting of Angpt2 has been implicated as a promising strategy for cancer treatment. Whereas miRNAs are emerging as important modulators of angiogenesis, regulation of Angpt2 by miRNAs has not been established. Here we firstly report that Ang2 is targeted by a microRNA, miRNA‐542‐3p, which inhibits tumour progression by impairing Ang2s pro‐angiogenic activity. In cultured endothelial cells, miR‐542‐3p inhibited translation of Angpt2 mRNA by binding to its 3′ UTR, and addition of miR‐542‐3p to cultured endothelial cells attenuated angiogenesis. Administration of miR‐542‐3p to tumour‐bearing mice reduced tumour growth, angiogenesis and metastasis. Furthermore, the level of miR‐542‐3p in primary breast carcinomas correlated inversely with clinical progression in primary tumour samples from stage III and IV patients. Together, these findings uncover a novel regulatory pathway whereby an anti‐angiogenic miR‐542‐3p directly targets the key angiogenesis‐promoting protein Angpt2, suggesting that miR‐542‐3p may represent a promising target for anti‐angiogenic therapy and a potential marker for monitoring disease progression. Copyright

The miR-30 Family Inhibits Pulmonary Vascular Hyperpermeability in the Premetastatic Phase by Direct Targeting of Skp2

Purpose: Before metastasis, primary tumor can create a premetastatic niche in distant organ to facilitate the dissemination of tumor cells. In the premetastatic phase, the permeability of pulmonary vasculatures is increased to accelerate the extravasation of circulating tumor cells. However, it is not clear whether local miRNAs contribute to the vascular hyperpermeability of the premetastatic niche. Experimental Design: The expression of total miRNAs was determined using microarray in series of premetastatic lungs from tumor-bearing mice. Significantly differentially expressed miRNAs were identified and validated with qRT-PCR. Vascular permeability assays, vascular mimic systems, and orthotopic tumor models were used to investigate roles of selected miRNAs and target genes in premetastatic hyperpermeability. Results: We identified a miRNA signature in premetastatic lungs. Among these miRNAs, miR-30a, b, c, d, and e were significantly attenuated. Subsequent investigations elucidated that lung fibroblast–derived miR-30s stabilized pulmonary vessels. Overexpression of miR-30s in lungs postponed metastasis and extended overall survival of B16 tumor–bearing mice. Following studies uncovered that Skp2 was directly targeted by miR-30s. Overexpression of Skp2 could disrupt pulmonary vessels, promote lung metastasis, and decrease overall survival of B16 tumor–bearing mice. Conclusions: These findings illuminate a novel mechanism for the modulation of premetastatic niches by miR-30s, which suggest that miR-30s represent not only promising targets for antimetastasis therapy but also indicators for metastasis. Clin Cancer Res; 21(13); 3071–80. ©2015 AACR.

The regulatory mechanism of a client kinase controlling its own release from Hsp90 chaperone machinery through phosphorylation

It is believed that the stability and activity of client proteins are passively regulated by the Hsp90 (heat-shock protein 90) chaperone machinery, which is known to be modulated by its intrinsic ATPase activity, co-chaperones and post-translational modifications. However, it is unclear whether client proteins themselves participate in regulation of the chaperoning process. The present study is the first example to show that a client kinase directly regulates Hsp90 activity, which is a novel level of regulation for the Hsp90 chaperone machinery. First, we prove that PKCγ (protein kinase Cγ) is a client protein of Hsp90α, and, that by interacting with PKCγ, Hsp90α prevents PKCγ degradation and facilitates its cytosol-to-membrane translocation and activation. A threonine residue set, Thr115/Thr425/Thr603, of Hsp90α is specifically phosphorylated by PKCγ, and, more interestingly, this threonine residue set serves as a ‘phosphorylation switch’ for Hsp90α binding or release of PKCγ. Moreover, phosphorylation of Hsp90α by PKCγ decreases the binding affinity of Hsp90α towards ATP and co-chaperones such as Cdc37 (cell-division cycle 37), thereby decreasing its chaperone activity. Further investigation demonstrated that the reciprocal regulation of Hsp90α and PKCγ plays a critical role in cancer cells, and that simultaneous inhibition of PKCγ and Hsp90α synergistically prevents cell migration and promotes apoptosis in cancer cells.

Endostatin Has ATPase Activity, Which Mediates Its Antiangiogenic and Antitumor Activities

Endostatin is an endogenous angiogenesis inhibitor with broad-spectrum antitumor activities. Although the molecular mechanisms of endostatin have been extensively explored, the intrinsic biochemical characteristics of endostatin are not completely understood. Here, we revealed for the first time that endostatin embedded novel ATPase activity. Moreover, mutagenesis study showed that the ATPase activity of endostatin mutants positively correlated with effects on endothelial cell activities and tumor growth. E-M, an endostatin mutant with higher ATPase activity than that of wild-type (WT) endostatin, significantly increased endostatin-mediated inhibitory effects on endothelial cell proliferation, migration, tube formation, and adhesion. In vivo study showed that E-M displayed enhanced antitumor effects compared with WT. On the other hand, K96A, K96R, and E176A, endostatin mutants with lower ATPase activities than that of WT, showed reduced or comparable effects on targeting both in vitro endothelial cell activities and in vivo tumor angiogenesis and tumor growth. Furthermore, endostatin and its mutants exhibited distinct abilities in regulations of gene expression (Id1, Id3), cell signaling (Erk, p38, and Src phosphorylation), and intracellular ATP levels. Collectively, our study demonstrates that endostatin has novel ATPase activity, which mediates its antiangiogenic and antitumor activities, suggesting that construction of endostatin analogues with high ATPase activity may provide a new direction for the development of more potent antiangiogenic drugs. Mol Cancer Ther; 14(5); 1192–201. ©2015 AACR.

Thr90 phosphorylation of Hsp90α by protein kinase A regulates its chaperone machinery.

Hsp90 (heat-shock protein 90) is one of the most important molecular chaperones in eukaryotes. Hsp90 facilitates the maturation, activation or degradation of its client proteins. It is now well accepted that both ATP binding and co-chaperone association are involved in regulating the Hsp90 chaperone machinery. However, other factors such as post-translational modifications are becoming increasingly recognized as being involved in this process. Recent studies have reported that phosphorylation of Hsp90 plays an unanticipated role in this process. In the present study, we systematically investigated the impact of phosphorylation of a single residue (Thr90) of Hsp90α (pThr90-Hsp90α) on its chaperone machinery. We demonstrate that protein kinase A specifically phosphorylates Hsp90α at Thr90, and that the pThr9090-Hsp90α level is significantly elevated in proliferating cells. Thr90 phosphorylation affects the binding affinity of Hsp90α to ATP. Subsequent examination of the interactions of Hsp90α with co-chaperones reveals that Thr90 phosphorylation specifically regulates the association of a subset of co-chaperones with Hsp90α. The Hsp90α T90E phosphor-mimic mutant exhibits increased association with Aha1 (activator of Hsp90 ATPase homologue 1), p23, PP5 (protein phosphatase 5) and CHIP (C-terminus of Hsp70-interacting protein), and decreased binding affinity with Hsp70, Cdc37 (cell division cycle 37) and Hop [Hsc70 (heat-shock cognate protein 70)/Hsp90-organizing protein], whereas its interaction with FKBP52 (FK506-binding protein 4) is only moderately affected. Moreover, we find that the ability of the T90E mutant to form complexes with its clients, such as Src, Akt or PKCγ (protein kinase Cγ), is dramatically impaired, suggesting that phosphorylation affects its chaperoning activity. Taken together, the results of the present study demonstrate that Thr90 phosphorylation is actively engaged in the regulation of the Hsp90α chaperone machinery and should be a generic determinant for the cycling of Hsp90α chaperone function.

Tumor cell-secreted angiogenin induces angiogenic activity of endothelial cells by suppressing miR-542-3p.

Therapeutic strategies for targeting angiogenesis have been proven as successful treatments for divergent cancers. We previously discovered an anti-angiogenic miR-542-3p, which directly targeted the key angiogenesis-promoting protein Angiopoietin-2 to inhibit tumor angiogenesis in breast cancer models. In this study, to further investigate the mechanism of miR-542-3p induced angiogenic inhibition, we screened for tumor cell derived factors which were responsible for miR-542-3p alteration in endothelial cells. We found that tumor cell-derived angiogenin downregulated miR-542-3p in endothelial cells. Overexpression of angiogenin in tumor cells facilitated angiogenic activation in both in vitro and in vivo models via inhibition of miR-542-3p. Furthermore, our results showed that angiogenin could suppress CEBPB and POU2F1, which were transcription factors for miR-542-3p, suggesting a novel tumor cell-endothelial cell signal pathway. In addition, the level of angiogenin in primary breast carcinomas correlated with clinical progression. Serum levels of angiogenin were associated with metastatic development of breast cancer patients. Together, these findings reveal a novel regulatory pathway whereby tumor-derived angiogenin directly activates angiogenesis through inhibition of miR-542-3p, suggesting that angiogenin may represent a promising target for anti-angiogenic therapy and a potential marker for monitoring disease progression.

Endostatin sensitizes p53-deficient non-small-cell lung cancer to genotoxic chemotherapy by targeting DNA-dependent protein kinase catalytic subunit

Endostatin was discovered as an endogenous angiogenesis inhibitor with broad‐spectrum antitumour activities. Although clinical efficacy was observed when endostatin was combined with standard chemotherapy for non‐small cell lung cancer (NSCLC), as well as other cancer types, the specific mechanisms underlying the benefit of endostatin are not completely understood. Extensive investigations suggest that endostatin is a multifunctional protein possessing more than anti‐angiogenic activity. Here, we found that endostatin exerts a direct chemosensitizing effect on p53‐deficient tumour cells. Concomitant treatment with endostatin and genotoxic drugs resulted in therapeutic synergy in both cellular and animal models of p53‐deficient NSCLC. Mechanistically, endostatin specifically interacts with DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in tumour cells and suppresses its DNA repair activity. Using isogenic NSCLC cells with different p53 statuses, we discovered that p53‐deficient tumour cells show chemoresistance to genotoxic drugs, creating a synthetic dependence on DNA‐PKcs‐mediated DNA repair. In this setting, endostatin exerted inhibitory effects on DNA‐PKcs activity, leading to accumulation of DNA lesions and promotion of the therapeutic effect of genotoxic chemotherapy. In contrast, p53‐proficient tumour cells were more sensitive to genotoxic drugs so that DNA‐PKcs could be cleaved by drug‐activated caspase‐3, making DNA‐PKcs inhibition less effective during this ongoing apoptotic process. Therefore, our data demonstrate a novel mechanism for endostatin as a DNA‐PKcs suppressor, and indicate that combination therapy of endostatin with genotoxic drugs could be a promising treatment strategy for cancer patients with p53‐deficient tumours. Copyright

Specific chemotherapeutic agents induce metastatic behaviour through stromal- and tumour-derived cytokine and angiogenic factor signalling

Recent studies reveal that chemotherapy can enhance metastasis due to host responses, such as augmented expression of adhesion molecules in endothelial cells and increased populations of myeloid cells. However, it is still unclear how tumour cells contribute to this process. Here, we observed that paclitaxel and carboplatin accelerated lung metastasis in tumour‐bearing mice, while doxorubicin and fluorouracil did not. Mechanistically, paclitaxel and carboplatin induced similar changes in cytokine and angiogenic factors. Increased levels of CXCR2, CXCR4, S1P/S1PR1, PlGF and PDGF‐BB were identified in the serum or primary tumour tissues of tumour‐bearing mice treated by paclitaxel. The serum levels of CXCL1 and PDGF‐BB and the tissue level of CXCR4 were also elevated by carboplatin. On the other hand, doxorubicin and fluorouracil did not induce such changes. The chemotherapy‐induced cytokine and angiogenic factor changes were also confirmed in gene expression datasets from human patients following chemotherapy treatment. These chemotherapy‐enhanced cytokines and angiogenic factors further induced angiogenesis, destabilized vascular integrity, recruited BMDCs to metastatic organs and mediated the proliferation, migration and epithelial‐to‐mesenchymal transition of tumour cells. Interestingly, inhibitors of these factors counteracted chemotherapy‐enhanced metastasis in both tumour‐bearing mice and normal mice injected intravenously with B16F10–GFP cells. In particular, blockade of the SDF‐1α–CXCR4 or S1P–S1PR1 axes not only compromised chemotherapy‐induced metastasis but also prolonged the median survival time by 33.9% and 40.3%, respectively. The current study delineates the mechanism of chemotherapy‐induced metastasis and provides novel therapeutic strategies to counterbalance pro‐metastatic effects of chemo‐drugs via combination treatment with anti‐cytokine/anti‐angiogenic therapy. Copyright

Endostatin Sensitizes p53‐Deficient Non‐Small Cell Lung Cancer to Genotoxic Chemotherapy by Targeting DNA‐PKcs

Endostatin was discovered as an endogenous angiogenesis inhibitor with broad‐spectrum antitumour activities. Although clinical efficacy was observed when endostatin was combined with standard chemotherapy for non‐small cell lung cancer (NSCLC), as well as other cancer types, the specific mechanisms underlying the benefit of endostatin are not completely understood. Extensive investigations suggest that endostatin is a multifunctional protein possessing more than anti‐angiogenic activity. Here, we found that endostatin exerts a direct chemosensitizing effect on p53‐deficient tumour cells. Concomitant treatment with endostatin and genotoxic drugs resulted in therapeutic synergy in both cellular and animal models of p53‐deficient NSCLC. Mechanistically, endostatin specifically interacts with DNA‐dependent protein kinase catalytic subunit (DNA‐PKcs) in tumour cells and suppresses its DNA repair activity. Using isogenic NSCLC cells with different p53 statuses, we discovered that p53‐deficient tumour cells show chemoresistance to genotoxic drugs, creating a synthetic dependence on DNA‐PKcs‐mediated DNA repair. In this setting, endostatin exerted inhibitory effects on DNA‐PKcs activity, leading to accumulation of DNA lesions and promotion of the therapeutic effect of genotoxic chemotherapy. In contrast, p53‐proficient tumour cells were more sensitive to genotoxic drugs so that DNA‐PKcs could be cleaved by drug‐activated caspase‐3, making DNA‐PKcs inhibition less effective during this ongoing apoptotic process. Therefore, our data demonstrate a novel mechanism for endostatin as a DNA‐PKcs suppressor, and indicate that combination therapy of endostatin with genotoxic drugs could be a promising treatment strategy for cancer patients with p53‐deficient tumours. Copyright