Increase of anti-metastatic efficacy by selectivity- but not affinity-optimization of synthetic serine protease inhibitors

Although tumors frequently show elevated protease activities, the concept of anti-proteolytic cancer therapy has lost momentum after failure of clinical trials with broad-spectrum matrix metalloproteinase inhibitors. Thus we need to adapt our design strategies for protease inhibitors. Here, we employed a series of seven structurally fine-modulated and pharmacokinetically closely related synthetic 4-amidinobenzylamine-based inhibitors with distinct selectivity for prototypical serine proteases in a murine T cell lymphoma liver metastasis model. This in vivo screening revealed efficacy of urokinase inhibitors but no correlation between urokinase selectivity or affinity and anti-metastatic effect. In contrast, factor Xa-selective inhibitors were more potent, demonstrating factor Xa or a factor Xa-like serine protease likely to be more determinant in this model. Factor Xa selectivity, but not affinity, significantly improved anti-metastatic efficacy. For example, factor Xa inhibitors CJ-504 and CJ-510 exert similar affinity for factor Xa (K(i)=14 nM versus 8.8 nM) but CJ-504 was 70-fold more selective for factor Xa. This correlated with higher anti-metastatic efficacy (58.8% with CJ-504; 28.2% with CJ-510). Our results show that among the protease inhibitors employed that have affinities in the nanomolar range, the strategy of selectivity-optimization is superior to further improvement of affinity to significantly enhance anti-metastatic efficacy. This appreciation may be important for the future rational design of new anti-proteolytic agents for cancer therapy.     

Apatinib for heavily treated patients with non-small cell lung cancer: Report of a case series and literature review

Although many strategies have been developed for non-small cell lung cancer (NSCLC), more secondary and further treatments are needed due to drug resistance or tumor recurrence. Apatinib is a novel oral antiangiogenic agent and in this study, we aim to investigate the clinical value of apatinib in heavily pretreated NSCLC. Here, we reported the characteristics, efficacy and adverse events of three patients treated with apatinib (500?mg/day). We also summarized the currently available evidence and ongoing clinical trials regarding the use of apatinib in NSCLC. Two cases of adenocarcinoma and one case of squamous cell carcinoma were treated with apatinib due to disease progression after previous treatments of chemotherapy and epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI). All patients responded to apatinib rapidly and underwent drug resistance shortly afterwards. The patient with squamous cell carcinoma died of hemoptysis. Other adverse events were acceptable. All previous relevant studies were compared and showed similar results but a longer progression-free survival. Additionally, ongoing clinical trials were systematically searched and listed. In conclusion, apatinib shows some efficacy in heavily treated NSCLC and generally tolerable toxicity in non-squamous NSCLC. More solid evidence will be accessible in near future.     

Effect of PKC412, a selective inhibitor of protein kinase C,on lung metastasis in mice injected with B16 melanoma cells

PKC412, a selective inhibitor of protein kinase C (PKC), is currently in clinical trials as an anti-tumor drug. In the present study, we investigated the anti-metastatic effect of PKC412 using an experimental metastatic mouse model intravenously injected with melanoma cells. One-hour exposure to various concentrations of PKC412 (0.5, 5 and 50 microM) dose-dependently reduced the lung-metastatic potential of highly metastatic B16-F10 and -BL6 mouse melanoma cells in syngeneic mice. Following the exposure, PKC activities in B16-F10 and -BL6 cells were significantly decreased, but growth curves were not influenced. To elucidate the mechanism of the anti-metastatic effect of PKC412, we examined the activity to invade the extracellular matrix and the platelet-aggregating activity of the melanoma cells incubated with PKC412 (0.5, 5 and 50 microM) for 1 hour. PKC412 significantly reduced both the invasive and platelet-aggregating activities. These results suggest that PKC412 shows an anti-metastatic function through the inhibition of the invasive and/or platelet-aggregating activities of melanoma cells. PKC412 is potentially a promising candidate for an anti-metastatic agent.     

Suppression of metastases of small cell lung cancer cells in mice by a peptidic CXCR4 inhibitor TF14016

CXCL12 is a chemokine essential for the organ-specific spread of a variety of cancers including small cell lung cancer (SCLC). Here, we examined the anti-metastatic efficacy of TF14016, a small peptidic inhibitor of CXCL12 receptor CXCR4, in SCLC. Treatment of mice with TF14016 significantly suppressed pulmonary metastases of CXCR4-expressing SCLC in size and number. Furthermore, histological examination revealed that the expression of vascular endothelial cell growth factor and the density of CD31-positive microvessels in metastatic foci were both significantly reduced in TF14016-treated mice. Collectively, CXCR4 could be an attractive target for anti-metastatic and anti-angiogenic therapy in SCLC.     

Inhibiting tissue invasion and metastasis as targets for cancer therapy

Many of the steps involved in cancer spread are potential targets for anti-metastatic treatment. Until recently, research aimed at inhibiting metastasis has concentrated on the proteases, especially on urokinase-type plasminogen activator and collagenase IV However, recent data suggests that both adhesion proteins and motility factors could also serve as targets for new treatments to prevent cancer invasion and metastasis. Almost all the work to date using anti-metastatic agents has been carried out using either in vitro artificial membranes or with animal models. It is, however, likely that some of the inhibitors of experimental metastasis which are described will be evaluated in clinical trials in the near future.     

Baicalein Inhibits Progression of Gallbladder Cancer Cells by Downregulating ZFX

Baicalein, a widely used Chinese herbal medicine, has multiple pharmacological activities. However, the precise mechanisms of the anti-proliferation and anti-metastatic effects of baicalein on gallbladder cancer (GBC) remain poorly understood. Therefore, the aim of this study was to assess the anti-proliferation and anti-metastatic effects of baicalein and the related mechanism(s) on GBC. In the present study, we found that treatment with baicalein induced a significant inhibitory effect on proliferation and promoted apoptosis in GBC-SD and SGC996 cells, two widely used gallbladder cancer cell lines. Additionally, treatment with baicalein inhibited the metastasis of GBC cells. Moreover, we demonstrated for the first time that baicalein inhibited GBC cell growth and metastasis via down-regulation of the expression level of Zinc finger protein X-linked (ZFX). In conclusion, our studies suggest that baicalein may be a potential phytochemical flavonoid for therapeutics of GBC and ZFX may serve as a molecular marker or predictive target for GBC.     

Curcumin inhibits hypoxia-induced migration in K1 papillary thyroid cancer cells

Curcumin, traditionally used as food and medicinal purposes, has recently been reported to have protective efficacy against hypoxia. Hypoxia is one of the important reactive factors in tumor metastasis, which is a key problem in clinical thyroid cancer therapy. In present study, we investigate the anti-metastatic effect of curcumin on the K1 papillary thyroid cancer cells as well as its potential mechanisms. The results show that curcumin effectively inhibits hypoxia-induced reactive oxygen species (ROS) upregulation and significantly decreases the mRNA and protein expression levels of hypoxia-inducible factor-1α (HIF-1α) in K1 cells. Curcumin also decreases the DNA binding ability of HIF-1α to hypoxia response element (HRE). Furthermore, curcumin enhances E-cadherin expression, inhibits metalloproteinase-9 (MMP-9) enzyme activity, and weakens K1 cells migration under hypoxic conditions. In summary, these results indicate that curcumin possesses a potent anti-metastatic effect and might be an effective tumoristatic agent for the treatment of aggressive papillary thyroid cancers.     

Metastasis of Breast Tumor Cells to Brain Is Suppressed by Phenethyl Isothiocyanate in a Novel In Vivo Metastasis Model

Breast tumor metastasis is a leading cause of cancer-related deaths worldwide. Breast tumor cells frequently metastasize to brain and initiate severe therapeutic complications. The chances of brain metastasis are further elevated in patients with HER2 overexpression. In the current study, we evaluated the anti-metastatic effects of phenethyl isothiocyanate (PEITC) in a novel murine model of breast tumor metastasis. The MDA-MB-231-BR (BR-brain seeking) breast tumor cells stably transfected with luciferase were injected into the left ventricle of mouse heart and the migration of cells to brain was monitored using a non-invasive IVIS bio-luminescent imaging system. In order to study the efficacy of PEITC in preventing the number of tumor cells migrating to brain, mice were given 10 µmol PEITC by oral gavage for ten days prior to intra-cardiac injection of tumor cells labeled with quantum dots. To evaluate the tumor growth suppressive effects, 10 µmol PEITC was given to mice every day starting 14^th day after intra-cardiac cell injection. Based on the presence of quantum dots in the brain section of control and treated mice, our results reveal that PEITC significantly prevented the metastasis of breast cancer cells to brain. Our results demonstrate that the growth of metastatic brain tumors in PEITC treated mice was about 50% less than that of control. According to Kaplan Meir’s curve, median survival of tumor bearing mice treated with PEITC was prolonged by 20.5%. Furthermore as compared to controls, we observed reduced HER2, EGFR and VEGF expression in the brain sections of PEITC treated mice. To the best of our knowledge, our study for the first time demonstrates the anti-metastatic effects of PEITC in vivo in a novel breast tumor metastasis model and provides the rationale for further clinical investigation.     

Anti-metastatic activity of hapten-modified autologous tumor cell vaccine in an animal tumor model

We used mice from which the primary 410.4 mammary carcinoma had been surgically excised to assess the anti-metastatic activity of low-dose cyclophosphamide (CY) followed by vaccination with dinitrophenyl (DNP)-modified, irradiated, autologous tumor cells (ATC) admixed with bacille Calmette-Guérin (BCG). Our studies revealed that CY treatment of mice followed by vaccination with DNP-modified. irradiated, ATC admixed with BCG improved the relapse-free survival compared to the survival of mice receiving either CY followed by vaccination with unmodified, irradiated, ATC admixed with BCG, or saline (control group). In addition, our studies demonstrated the importance of CY administration in eliciting the therapeutic effect of DNP-modified ATC vaccine against metastatic disease. The therapeutic effect of CY followed by DNP-modified ATC vaccine was abrogated by depletion of CD4(+) or CD8(+) T-cells, illustrating the importance of both T-cell subsets for the anti-metastatic effect of this therapeutic protocol. In addition, neutralizing anti-IFN-gamma monoclonal antibody (mAb), or neutralizing anti-tumor necrosis factor (TNF) mAb reduced the relapse-free survival of mice treated with CY followed by DNP-modified ATC vaccine, indicating the importance of both cytokines for the realization of the anti-metastatic effect of this therapeutic protocol. Since the therapeutic protocol used in our studies was similar to that employed by Berd et al. as postsurgical adjuvant therapy in cancer patients and yielded a comparable anti-metastatic effect, the information obtained from the current studies with our clinically relevant experimental tumor model is expected to shed light on the mechanism(s) by which the anti-metastatic effect of this post-surgical adjuvant therapy is realized in cancer patients.     

Accelerated LV remodeling after myocardial infarction in TIMP-1-deficient mice: effects of exogenous MMP inhibition

Alterations in matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) have been implicated in adverse left ventricular (LV) remodeling after myocardial infarction (MI). However, the direct mechanistic role of TIMPs in the post-MI remodeling process has not been completely established. The goal of this project was to define the effects of altering endogenous MMP inhibitory control through combined genetic and pharmacological approaches on post-MI remodeling in mice. This study examined the effects of MMP inhibition (MMPi) with PD-166793 (30 mg.kg(-1).day(-1)) on LV geometry and function (conductance volumetry) after MI in wild-type (WT) mice and mice deficient in the TIMP-1 gene [TIMP-1 knockout (TIMP1-KO)]. At 3 days after MI (coronary ligation), mice were randomized into four groups: WT-MI/MMPi (n = 10), TIMP1-KO-MI/MMPi (n = 10), WT-MI (n = 22), and TIMP1-KO-MI (n = 23). LV end-diastolic volume (EDV) and ejection fraction were determined 14 days after MI. Age-matched WT (n = 20) and TIMP1-KO (n = 28) mice served as reference controls. LVEDV was similar under control conditions in WT and TIMP1-KO mice (36 +/- 2 and 40 +/- 2 microl, respectively) but was greater in TIMP1-KO-MI than in WT-MI mice (48 +/- 2 vs. 61 +/- 5 microl, P < 0.05). LVEDV was reduced from MI-only values in WT-MI/MMPi and TIMP1-KO-MI/MMPi mice (42 +/- 2 and 36 +/- 2 microl, respectively, P < 0.05) but was reduced to the greatest degree in TIMP1-KO mice (P < 0.05). LV ejection fraction was reduced in both groups after MI and increased in TIMP1-KO-MI/MMPi, but not in WT-MI/MMPi, mice. These unique results demonstrated that myocardial TIMP-1 plays a regulatory role in post-MI remodeling and that the accelerated myocardial remodeling induced by TIMP-1 gene deletion can be pharmacologically "rescued" by MMP inhibition. These results define the importance of local endogenous control of MMP activity with respect to regulating LV structure and function after MI.     

N-i-Propoxy-N-biphenylsulfonylaminobutylhydroxamic acids as potent and selective inhibitors of MMP-2 and MT1-MMP

Structural manipulation of the pharmacophoric model of type A selective MMP inhibitors (MMPi), obtained by the insertion of some alkyl substituents R2 possessing an appropriate geometry, steric bulkiness and lipophilicity, is able to improve potency, in the subnanomolar range on MMP-2, and to give a good MMP inhibition on MMP-14 (MT1-MMP) in the designed MMPi of type C, while maintaining a good MMP-1/MMP-2 selectivity profile. The simultaneous inhibition of these two enzymes yields type C compounds, which are potent antiangiogenic agents, able to block a chemoinvasion model on HUVEC cells in the micromolar range.     

Comparison of Serum and Tissue Levels of Trace Elements in Different Models of Cervical Cancer

Cervical cancer is a leading cause of death by cancer among women worldwide. It is necessary to develop and refine cervical cancer models to more accurately reflect human tumor type. The relevance of cervical cancer to trace element was studied in this paper. By means of quantitative trace element analysis in models and patients with cervical cancer, the tissue and serum levels of trace elements in papillomaviruses-induced cancer models were more similar to that of patients than the levels in models induced by HeLa cell and methylcholanthrene. The results reflect papillomaviruses model most accurately mimic in vivo carcinogenesis of patients with cervical cancer. It will have a superior predictive value over HeLa cell and methylcholanthrene models in pre-clinical trials. The papillomaviruses-induced cervical cancer can provide more reliable models for testing the efficacy of drugs in treating human cancers.     

A facile and sensitive method of quantifying glutaminase binding to its inhibitor CB-839 in tissues

Many cancer types reprogram their metabolism to become addicted to glutamine. One of the critical enzymes in the utilization of glutamine in these cells is glutaminase. CB-839 (telaglenastat) is a drug that targets glutaminase that is currently being evaluated in many clinical trials for efficacy in various cancer types that are known to be driven by glutamine metabolism. Despite its use, there are limited assays available for testing the pharmacodynamic on-target effects of CB-839 on the limited, small-volume patient samples that are obtained in early-phase clinical trials. Thus, we developed an assay based on the cellular thermal shift assay technique using AlphaLISA technology to show that CB-839 specifically engages glutaminase in colon cancer cell lines in vitro and in minute quantities of mouse xenograft tumors. Notably, we show that this assay detects CB-839 binding to glutaminase in platelets of patients collected while receiving CB-839 on a clinical trial. This assay may be used to study the pharmacodynamic profile of CB-839 in very small tissue samples obtained from patients on a clinical trial and may be useful in future studies designed to screen other inhibitors of glutaminase.     

MMP inhibitors augment fibroblast adhesion through stabilization of focal adhesion contacts and up-regulation of cadherin function

Increased pericellular proteolysis due to an imbalance between MMPs (matrix metalloproteinases) and TIMPs (tissue inhibitors of metalloproteinases) promotes early stages of tumorigenesis. We have reported that TIMP-1 down-regulation confers tumorigenicity on immortal Swiss 3T3 fibroblasts. In pursuit of the mechanism involved in this transformation, we asked whether MMP inhibitors modulate contact inhibition and cell adhesion, because the dysregulation of these events is essential for cellular transformation. Using both genetic and biochemical means, we demonstrate that MMP inhibitors regulate fibroblast cell adhesion. TIMP-1 down-regulated cells formed dense, multilayered colonies, suggesting a loss of contact inhibition. Recombinant TIMP-1 and synthetic MMP inhibitors (MMPi) restored normal cell contact and density of these cells in a dose-dependent manner. Consequently, the effect of MMPi on both cell-extracellular matrix (ECM) and cell-cell adhesion were investigated. Upon MMPi treatment, p125(FAK) was redistributed, together with vinculin, to points of cell-ECM contact. Furthermore, phosphorylation of p125(FAK) was restored to levels similar to that of wild type. In parallel, MMPi treatment increased cadherin levels and stabilized cadherin-mediated cell-cell contacts. Moreover, enhanced cadherin function was evident as increased calcium-dependent cell-cell aggregation and co-localization of cadherin and beta-catenin at the cell membrane. We also obtained independent evidence of altered cadherin function using timp-1(-/-) mouse embryonic fibroblasts. Our data provide provocative evidence that increased pericellular proteolysis impacts cell adhesion systems to offset normal contact inhibition, with subsequent effects on cell transformation and tumorigenesis.     

CAR-T Cell Therapy for Breast Cancer: From Basic Research to Clinical Application

Breast cancer rises as the most commonly diagnosed cancer in 2020. Among women, breast cancer ranks first in both cancer incidence rate and mortality. Treatment resistance developed from the current clinical therapies limits the efficacy of therapeutic outcomes, thus new treatment approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a type of immunotherapy developed from adoptive T cell transfer, which typically uses patients'' own immune cells to combat cancer. CAR-T cells are armed with specific antibodies to recognize antigens in self-tumor cells thus eliciting cytotoxic effects. In recent years, CAR-T cell therapy has achieved remarkable successes in treating hematologic malignancies; however, the therapeutic effects in solid tumors are not up to expectations including breast cancer. This review aims to discuss the development of CAR-T cell therapy in breast cancer from preclinical studies to ongoing clinical trials. Specifically, we summarize tumor-associated antigens in breast cancer, ongoing clinical trials, obstacles interfering with the therapeutic effects of CAR-T cell therapy, and discuss potential strategies to improve treatment efficacy. Overall, we hope our review provides a landscape view of recent progress for CAR-T cell therapy in breast cancer and ignites interest for further research directions.     

Inhibitory Activity of (+)-Usnic Acid against Non-Small Cell Lung Cancer Cell Motility

Lichens are symbiotic organisms that produce various unique chemicals that can be used for pharmaceutical purposes. With the aim of screening new anti-cancer agents that inhibit cancer cell motility, we tested the inhibitory activity of seven lichen species collected from the Romanian Carpathian Mountains against migration and invasion of human lung cancer cells and further investigated the molecular mechanisms underlying their anti-metastatic activity. Among them, Alectoria samentosa, Flavocetraria nivalis, Alectoria ochroleuca, and Usnea florida showed significant inhibitory activity against motility of human lung cancer cells. HPLC results showed that usnic acid is the main compound in these lichens, and (+)-usnic acid showed similar inhibitory activity that crude extract have. Mechanistically, β-catenin-mediated TOPFLASH activity and KITENIN-mediated AP-1 activity were decreased by (+)-usnic acid treatment in a dose-dependent manner. The quantitative real-time PCR data showed that (+)-usnic acid decreased the mRNA level of CD44, Cyclin D1 and c-myc, which are the downstream target genes of both β-catenin/LEF and c-jun/AP-1. Also, Rac1 and RhoA activities were decreased by treatment with (+)-usnic acid. Interestingly, higher inhibitory activity for cell invasion was observed when cells were treated with (+)-usnic acid and cetuximab. These results implied that (+)-usnic acid might have potential activity in inhibition of cancer cell metastasis, and (+)-usnic acid could be used for anti-cancer therapy with a distinct mechanisms of action.     

Tumor vaccines in 2010: Need for integration

Induction of tumor-specific immunity is an attractive approach to cancer therapy, however to date every major pivotal trial has resulted in failure. While the phenomena of tumor-mediated immune suppression has been known for decades, only recently have specific molecular pathways been elucidated, and for the first time, rationale means of intervening and observing results of intervention have been developed. In this review we describe major advances in our understanding of tumor escape from immunological pressure and provide some possible therapeutic scenarios for enhancement of efficacy in future cancer vaccine trials.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Baohuoside i suppresses breast cancer metastasis by downregulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 pathway

BackgroundBreast cancer is the most common malignancy in women and metastasis is the leading cause of breast cancer-related deaths. Our previous studies have shown that XIAOPI formula, a newly approved drug by the State Food and Drug Administration of China (SFDA), can dramatically inhibit breast cancer metastasis by modulating the tumor-associated macrophages/C-X-C motif chemokine ligand 1 (TAMs/CXCL1) pathway. However, the bioactive compound accounting for the anti-metastatic effect of XIAOPI formula remains unclear.PurposeThis study was designed to separate the anti-metastatic bioactive compound from XIAOPI formula and to elucidate its action mechanisms.Study Design/MethodsTAMs/CXCL1 promoter activity-guided fractionation and multiple chemical structure identification approaches were conducted to screen the bioactive compound from XIAOPI formula. Breast cancer cells and TAMs were co-cultured in vitro or co-injected in vivo to simulate their coexistence. Multiple molecular biology experiments, zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts were applied to validate the anti-metastatic activity of the screened compound.ResultsBioactivity-guided fractionation identified baohuoside I (BHS) as the key bioactive compound of XIAOPI formula in inhibiting TAMs/CXCL1 promoter activity. Functional studies revealed that BHS could significantly inhibit the migration and invasion as well as the expression of metastasis-related proteins in both human and mouse breast cancer cells, along with decreasing the proportion of breast cancer stem cells (CSCs). Furthermore, BHS could suppress the M2 phenotype polarization of TAMs and therefore attenuate their CXCL1 expression and secretion. Notably, mechanistic investigations validated TAMs/CXCL1 as the crucial target of BHS in suppressing breast cancer metastasis as exogenous addition of CXCL1 significantly abrogated the anti-metastatic effect of BHS on breast cancer cells. Moreover, BHS was highly safe in vivo as it exhibited no observable embryotoxicity or teratogenic effect on zebrafish embryos. More importantly, BHS remarkably suppressed breast cancer metastasis and TAMs/CXCL1 activity in both zebrafish breast cancer xenotransplantation model and mouse breast cancer xenografts.ConclusionThis study not only provides novel insights into TAMs/CXCL1 as a reliable screening target for anti-metastatic drug discovery, but also suggests BHS as a promising candidate drug for metastatic breast cancer treatment.