Role of Cyclin D1b in Inducing Macrophages Toward a Tumor-associated Macrophage-like Phenotype in Murine Breast Cancer.

OBJECTIVE: Tumor-associated macrophages (TAMs) of the M2 phenotype are frequently associated with cancer progression. Invasive cancer cells undergoing epithelial-mesenchymal transition (EMT) have a selective advantage as TAM activators. Cyclin D1b is a highly oncogenic splice variant of cyclin D1. We previously reported that cyclin D1b enhances the invasiveness of breast cancer cells by inducing EMT. However, the role of cyclin D1b in inducing macrophage differentiation toward tumor-associated macrophage-like cells remains unknown. This study aimed to explore the relationship between breast cancer cells overexpressing cyclin D1b and TAMs. METHODS: Mouse breast cancer 4T1 cells were transfected with cyclin D1b variant and co-cultured with macrophage cells in a Transwell coculture system. The expression of characteristic cytokines in differentiated macrophages was detected using qRT-PCR, ELISA and zymography assay. Tumor-associated macrophage distribution in a transplanted tumor was detected by immunofluorescence staining. The proliferation and migration ability of breast cancer cells was detected using the cell counting kit-8 (CCK-8) assay, wound healing assay, Transwell invasion assay, and lung metastasis assay. Expression levels of mRNAs were detected by qRT-PCR. Protein expression levels were detected by Western blotting. The integrated analyses of The Cancer Genome Atlas (TCGA) datasets and bioinformatics methods were adopted to discover gene expression, gene coexpression, and overall survival in patients with breast cancer. RESULTS: After co-culture with breast cancer cells overexpressing cyclin D1b, RAW264.7 macrophages were differentiated into an M2 phenotype. Moreover, differentiated M2-like macrophages promoted the proliferation and migration of breast cancer cells in turn. Notably, these macrophages facilitated the migration of breast cancer cells in vivo. Further investigations indicated that differentiated M2-like macrophages induced EMT of breast cancer cells accompanied with upregulation of TGF-ß1 and integrin ß3 expression. CONCLUSION: Breast cancer cells transfected with cyclin D1b can induce the differentiation of macrophages into a tumor-associated macrophage-like phenotype, which promotes tumor metastasis in vitro and in vivo.

The locoregional adiponectin and its synergistic antitumor effect with HIF-1α blockade in TSCC.

Adiponectin (APN) is a kind of endogenous anti-tumor adipocytokine, which exerts its function by binding to its receptors (AdipoR1 and AdipoR2). However, hyperadiponectinemia is found in some pathophysiological processes without significant protective effect, which indicates the existence of APN resistance. Here, we aimed to investigate the locoregional expression of APN in tongue squamous cell carcinoma (TSCC) tissues, and to explore the potential regulatory mechanism of APN resistance under hypoxia. Consequently, we found that the protein expression of APN and AdipoR1, but not AdipoR2, was upregulated in the early stage of TSCC and after hypoxic treatment ex vivo and in vitro. Knockdown of HIF-1α decreased the level of APN and AdipoR1, and simultaneously, HIF-1α was identified as transcriptor of the APN. Intriguingly, a regenerative feedback of HIF-1α was unexpectedly detected after application of recombinant globular APN (gAPN), which most likely contributed to the APN resistance. Furthermore, HIF-1α blockade combined with gAPN has a prominent synergistic antitumor effect, which suggested an effective amelioration in APN resistance. In all, our study revealed the possible mechanism of APN resistance under hypoxia and provides a promising strategy of bi-target treatment with APN and HIF-1α for TSCC therapy.

Cyclin D1b Splice Variant Promotes αvß3-mediated EMT Induced by LPS in Breast Cancer Cells.

Epithelial-to-mesenchymal transition (EMT) plays a critical role in cancer metastasis, and is relevant to the inflammatory microenvironment. Lipopolysaccharide (LPS), a cell wall constituent of gram-negative bacteria, has been reported to induce EMT of cancer cells through TLR4 signal. We previously reported that LPS promoted metastasis of mesenchymallike breast cancer cells with high expression of cyclin D1b. However, the role of cyclin D1b in LPS-induced EMT has not been fully elucidated. In the present study, we described that cyclin D1b augmented EMT induced by LPS in MCF-7 breast cancer cells. Cyclin D1b markedly amplified integrin αvß3 expression, which was further up-regulated under LPS stimulation. Our results showed ectopic expression of cyclin D1b promoted invasiveness of epithelial-like MCF-7 cells under LPS stimulation. Additionally, LPS-induced metastasis and EMT in MCF-7-D1b cells might depend on αvß3 expression. Further exploration indicated that cyclin D1b cooperated with HoxD3, a transcription factor promoting αvß3 expression, to promote LPSinduced EMT. Knockout of HoxD3 repressed LPS-induced EMT and αvß3 over-expression in MCF-7 cells with high expression of cyclin D1b. Specifically, all these effects were in a cyclin Dla independent manner. Taken all together, LPS up-regulated integrin αvß3 expression in MCF-7 cells with high expression of cyclin D1b and induced EMT in breast cancer cells, which highlights that cyclin D1b may act as an endogenous pathway participating in exogenous signal inducing EMT in breast cancer cells.

Characterization and functional analysis of a slow-cycling subpopulation in colorectal cancer enriched by cell cycle inducer combined chemotherapy.

The concept of cancer stem cells has been proposed in various malignancies including colorectal cancer. Recent studies show direct evidence for quiescence slow-cycling cells playing a role in cancer stem cells. There exists an urgent need to isolate and better characterize these slow-cycling cells. In this study, we developed a new model to enrich slow-cycling tumor cells using cell-cycle inducer combined with cell cycle-dependent chemotherapy in vitro and in vivo. Our results show that Short-term exposure of colorectal cancer cells to chemotherapy combined with cell-cycle inducer enriches for a cell-cycle quiescent tumor cell population. Specifically, these slow-cycling tumor cells exhibit increased chemotherapy resistance in vitro and tumorigenicity in vivo. Notably, these cells are stem-cell like and participate in metastatic dormancy. Further exploration indicates that slow-cycling colorectal cancer cells in our model are less sensitive to cytokine-induced-killer cell mediated cytotoxic killing in vivo and in vitro. Collectively, our cell cycle inducer combined chemotherapy exposure model enriches for a slow-cycling, dormant, chemo-resistant tumor cell sub-population that are resistant to cytokine induced killer cell based immunotherapy. Studying unique signaling pathways in dormant tumor cells enriched by cell cycle inducer combined chemotherapy treatment is expected to identify novel therapeutic targets for preventing tumor recurrence.

Triterpenoid saponin flaccidoside II from Anemone flaccida triggers apoptosis of NF1-associated malignant peripheral nerve sheath tumors via the MAPK-HO-1 pathway.

Malignant peripheral nerve sheath tumors (MPNSTs) are highly aggressive soft tissue neoplasms that are extremely rare and are frequently associated with neurofibromatosis type 1 patients. MPNSTs are typically fatal, and there is no effective treatment so far. In our previous study, we showed that flaccidoside II, one of the triterpenoid saponins isolated from Anemone flaccida Fr. Schmidt, has antitumor potential by inducing apoptosis. In the present study, we found that flaccidoside II inhibits proliferation and facilitates apoptosis in MPNST cell lines ST88-14 and S462. Furthermore, this study provides a mechanism by which the downregulation of heme oxygenase-1 via extracellular signal-regulated kinase-1/2 and p38 mitogen-activated protein kinase pathways is involved in the apoptotic role of flaccidoside II. This study suggested the potential of flaccidoside II as a novel pharmacotherapeutic approach for MPNSTs.

Cyclin D1b splice variant promotes αvß3-mediated adhesion and invasive migration of breast cancer cells.

Cyclin D1b, a splice variant of the cell cycle regulator cyclin D1, holds oncogenic functions in human cancer. However, the mechanisms underlying cyclin D1b function remain poorly understood. Here we introduced wild-type cyclin D1a or cyclin D1b variant into non-metastatic MCF-7 cells. Our results show that ectopic expression of cyclin D1b promotes invasiveness of the cancer cells in a cyclin D1a independent manner. Specifically, cyclin D1b is found to modulate the expression of αvß3, which characterizes the metastatic phenotype, and enhance tumor cell invasive potential in cooperating with HoxD3. Notably, cyclin D1b promotes αvß3-mediated adhesion and invasive migration, which are associated with invasive potential of breast cancer cells. Further exploration indicates that cyclin D1b makes breast cancer cells more sensitive to toll-like receptor 4 ligand released from damaged tumor cells. These findings reveal a role of cyclin D1b as a possible mediator of αvß3 transcription to promote tumor metastasis.

Extracellular HSPA1A promotes the growth of hepatocarcinoma by augmenting tumor cell proliferation and apoptosis-resistance.

Intracellular HSP70 has been implicated as a cytoprotective protein, whereas the effect of extracellular HSP70 on tumor cells has not been fully understood to date. Here we report that extracellular HSPA1A, a stress-inducible member of HSP70 family, could promote tumor growth. HSPA1A promoted the proliferation of H22 hepatocarcinoma cells through TLR2 and TLR4 signaling. The effect of HSPA1A was abolished by inhibiting NF-κB. HSPA1A also augmented the apoptosis-resistance of H22 cells by activating NF-κB, thus to promote the proliferation of H22 cells in presence of mitomycin C. Furthermore, the promoting effect of HSPA1A on tumor cell proliferation was existent after the removal of HSPA1A, which might involve HSPA1A-promoted upregulation of TLR4 expression in tumor cells and release of HMGB1 from tumor cells. These findings suggest that extracellular HSPA1A functions as endogenous ligand for TLR2 and TLR4 to facilitate tumor growth.

Triggering of Toll-like receptor 4 on metastatic breast cancer cells promotes αvß3-mediated adhesion and invasive migration.

Triggering of Toll-like receptor 4 (TLR4) on tumor cells has been found to promote tumor progression by promoting tumor cell proliferation and survival. So far, however, the effect of TLR4 signaling on tumor metastasis has not been well elucidated. Here, we report that triggering of TLR4 on metastatic breast cancer cells could reciprocally regulate the expression of αvß3 and the expressions of TPM1 and maspin, and promote αvß3-mediated adhesion and invasive migration of the cells. In metastatic breast cancer cells, TLR4 signaling increased the expression of integrin αvß3 by activating NF-κB, resulting in the increased adhesion capacity of tumor cells to the ligand for αvß3, and the increased polymerization of actin and production of MMP-9 in tumor cells in response to ECM. HoxD3 was required for the up-regulation of αv and ß3 expressions by NF-κB. Moreover, TLR4 signaling increased the expression of miR-21 in breast cancer cells by activating NF-κB. Accordingly, the expressions of TPM1 and maspin were decreased at protein level, whereas the transcription activity of these genes was not influenced. Consistent with the promoting effect on αvß3-mediated adhesion and invasive migration, TLR4 signaling promoted the arrest of metastatic breast cancer cells in circulation and following invasion. The effect of TLR4 signaling could be abrogated by inhibiting NF-κB. These findings suggest that metastatic breast cancer cells could acquire higher metastatic potential due to triggering of TLR4 and activation of NF-κB in the cells, and that both TLR4 and NF-κB could be therapeutic targets for preventing metastasis of breast cancer cells.

Endothelial cell-expressed Tim-3 facilitates metastasis of melanoma cells by activating the NF-kappaB pathway.

T cell immunoglobulin and mucin domain-3 (Tim-3) is originally recognized as a receptor of Th1 cells. We found that Tim-3 could be expressed in endothelial cells after stimulation with tumor cell-released TLR4 ligand. Tim-3 expressed by endothelial cells does not function as the receptor of galectin-9, but mediates the interaction of endothelial cells with tumor cells. The engagement of endothelial cell-expressed Tim-3 with a non-galectin 9 putative receptor on B16 melanoma cells could trigger the NF-kappaB signaling pathway in B16 cells. The activated NF-kappaB not only promoted the proliferation of B16 cells, but also enhanced apoptosis resistance of B16 cells by up-regulating Bcl-2 and Bcl-xL and down-regulating Bax. Consistently, Tim-3 facilitated the survival of B16 cells in the blood stream, arrested in the lung and following invasion, resulted in more metastatic nodules in the lung. These findings suggest that endothelial cell-expressed Tim-3 increases tumor cell metastatic potential by facilitating tumor cell intravasation, survival in blood stream and extravasation. Thus, anti-inflammation or blockade of Tim-3 may contribute to the prevention of metastasis.

Recombinant CBD-HepII polypeptide of fibronectin inhibits alphavbeta3 signaling and hematogenous metastasis of tumor.

The interaction of integrin alphavbeta3 and its ligands are crucial for tumor metastasis. Recombinant CBD-HepII polypeptide of fibronectin, designated as CH50, suppressed the binding of tumor cells to ECM molecules, and abolished the promoting effect of soluble fibronectin and fibrinogen on tumor cell adhesion to ECM molecules. The underlying mechanisms involve the blockade and downregulation of alphavbeta3 and its co-receptor syndecan 1 by CH50. The activation of FAK, upregulation of cdc2, the production and activation of MMP-2 and MMP-9 by ECM molecules-stimulated tumor cells were inhibited by CH50. CH50 reduced the tumor cell arrest during blood flow, and also inhibited the invasive ability of tumor cells. The in vivo expressed CH50 suppressed the lung metastasis of circulating tumor cells, and prolonged the survival of mice after tumor cell inoculation. These findings suggest a prospective utility of CH50 in the gene therapy for prevention of tumor metastasis.

Inhibition of hepatocarcinoma and tumor metastasis to liver by gene therapy with recombinant CBD-HepII polypeptide of fibronectin.

Unlike the intact fibronectin (FN) molecule, some proteolytic or recombinant fragments of FN possess inhibitory activities on tumor, providing potential strategies in tumor therapeutics. Using the hydrodynamics-based gene delivery technique, we demonstrated that the treatment by in vivo expression of a recombinant CBD-HepII polypeptide of FN, designated as CH50, strongly inhibited the tumor growth, tumor invasion and angiogenesis. Such inhibitory effects of CH50 on tumor were partly ascribed to its influence on the activities of MMP-9 and alphavbeta3 integrin. The in vivo expressed CH50 decreased both the production and the activity of MMP-9 in tumor tissues. CH50 also down-regulated alphavbeta3 expression in tumor cells and endothelial cells in vitro. The decreased activity of alphavbeta3 integrin was proved by its reduced binding ability to fibrinogen and the down-regulation of cdc2 expression. The gene therapy with CH50 not only prolonged the survival of mice bearing hepatocarcinoma in the liver, but also suppressed the growth and invasive ability of tumor in spleen and its metastasis to liver. Taken together, these findings suggest a prospective utility of CH50 in the gene therapy of liver cancer.

Soluble PD-1 facilitates 4-1BBL-triggered antitumor immunity against murine H22 hepatocarcinoma in vivo.

PURPOSE: The use of costimulatory molecules targeting distinct T-cell signaling pathways has provided a means for triggering and enhancing antitumor immunity; however, it is still not fully understood what types of costimulatory molecules are suitable for the combination in tumor therapy. Our purpose in this study is to establish an effective antitumor immune approach by using costimulatory molecule 4-1BBL in combination with soluble PD-1. EXPERIMENTAL DESIGN: The murine H22 hepatocarcinoma served as an ectopic tumor model. Local gene transfer was done by injection with naked plasmid p4-1BBL and/or psPD-1. The synergistic mechanism of dual-gene therapy was elucidated by detecting the change of gene expression of immunoregulatory factors in tumor microenvironment. The effects of immunotherapy were evaluated by testing the function of tumor-specific T cells, measuring tumor weight or volume, survival of mice, and H&E staining of tissues. RESULTS: 4-1BBL expressed by normal nonimmune cells effectively enhanced antitumor immune response but up-regulated PD-L1 and did not reduce IL-10 and transforming growth factor-beta (TGF-beta). sPD-1 synergized with 4-1BBL to establish efficient antitumor immune environment, including down-regulation of IL-10 and TGF-beta, further up-regulation of interleukin (IL)-2 and IFN-gamma, and higher CD8(+) T-cell infiltration. The combined treatment by 4-1BBL/sPD-1 eradicated tumors from mice with small amounts of preexistent tumor cells or tumors from approximately 60% of individuals with larger amounts of preexistent tumor cells. CONCLUSIONS: Our findings in this report imply a great potential of 4-1BBL in combination with sPD-1 in tumor therapeutics with the in vivo existent tumor cells as antigens.

[The inhibitory effect of recombinant polypeptide CH50 of fibronectin on invasion and angiogenesis of tumors].

OBJECTIVE: To investigate the inhibitory effect of recombinant polypeptide CH50 of fibronectin on invasion and angiogenesis of tumors, and analyze the possible molecular mechanism of the therapeutic effect of polypeptide CH50 on tumors. METHODS: The tumor model was established by inoculation of H22 hepatocarcinoma cells in mice. The tumor gene therapy was performed by in vivo gene transfection with a method based on hydrodynamics to express polypeptide CH50. After treatment, the inhibitory effect on tumor invasion and angiogenesis was observed by histotology with HE staining of tumor tissues. The expresison of MMP-9 mRNA and protein at the edge of tumor tissue was evaluated by RT-PCR and gelatin zymography, respectively. RT-PCR was used to detect the expression of the related genes in H22 cells treated with polypeptide CH50. Cell adhesion assay was used to analyze the influence of polypeptide CH50 on the binding of cells to fibrinogen. RESULTS: (1) Eukaryotic expression plasmid pCH510 was expressed in vivo in a non-targeting manner and produced a significant inhibitory effect on tumor growth. The therapy with polypeptide CH50 resulted in pronounced necrosis of tumor cells in pCH510 group, compared with that in control groups at histological level. (2) Polypeptide CH50 could inhibit the growth, invasion and angiogenesis of the tumor, and interfere the formation of new collateral circulation in the tumor. (3) The expression level of MMP-9 protein at the edge of tumor tissue was significantly decreased after treatment, especially the activation of pro-MMP-9 was inhibited significantly, whereas the expression level of MMP-9 mRNA was not influenced. (4) The expression of alphav, 33 and cdc2 mRNAs in H22 cells treated with polypeptide CH50 was down-regulated. (5) Cell adhesion assay manifested that polypeptide CH50 can affect the adhesion ability of H22 cells. CONCLUSION: Polypeptide CH50 can inhibit tumor growth and angiogenesis by suppressing the functions of MMP-9 and integrin alphavbeta3.