Journal of Experimental & Clinical Cancer Research

The α6β4 integrin is overexpressed in the basal subtype of breast cancer and plays an important role in tumor cell motility and invasion. EGFR is also overexpressed in the basal subtype of breast cancer, and crosstalk between α6β4 integrin and EGFR appears to be important in tumor progression. We evaluated the effects of α6β4 crosslinking on the distribution and function of EGFR in breast carcinoma cell line MDA-MB-231. Receptor distribution was evaluated by fluorescence microscopy and multispectral imaging flow cytometry, and ligand-mediated EGFR signaling was evaluated using Western blots and a Rho pull-down assay. Antibody-mediated crosslinking of α6β4 integrin was sufficient to induce cell-surface clustering of not only α6β4 but also EGFR in nonadherent cells. The induced clustering of EGFR was observed minimally after 5 min of integrin crosslinking but was more prominent after 15 min. EGFR clustering had minimal effect on the phosphorylation of Akt or Erk1,2 in response to EGF in suspended cells or in response to HB-EGF in adherent cells. However, EGFR clustering induced by crosslinking α6β4 had a marked effect on Rho activation in response to EGF. Crosslinking α6β4 integrin in breast carcinoma cells induces EGFR clustering and preferentially promotes Rho activation in response to EGF. We hypothesize that this integrin-EGFR crosstalk may facilitate tumor cell cytoskeletal rearrangements important for tumor progression.

Several reports have described the potential effects of natural compounds as anti-cancer agents in vitro as well as in vivo. The aim of this study was to evaluate the anti-cancer effect of Limoniastrum guyonianum aqueous gall extract (G extract) and luteolin in the human cervical cancer HeLa cell line, and, if so, to clarify the underlying mechanism. Our results show that G extract and luteolin inhibited cell proliferation and induced G2/M cell cycle arrest in a concentration and time-dependent manner. Both natural products induced programmed cell death as confirmed by the presence of hypodiploid G0/G1 cells. These effects are associated with an up-regulation of the expression of the tumor suppressor gene p16 ^INK4A and a down-regulation of the expression of the anti-apoptotic actor UHRF1 and its main partner DNMT1. Moreover, G extract- and luteolin-induced UHRF1 and DNMT1 down-regulation is accompanied with a global DNA hypomethylation in HeLa cell line. Altogether our results show that G extract mediates its growth inhibitory effects on human cervical cancer HeLa cell line likely via the activation of a p16^INK4A -dependent cell cycle checkpoint signalling pathway orchestrated by UHRF1 and DNMT1 down-regulation.

Về mặt lâm sàng, ung thư vú thường được phân loại thành hai kiểu phụ dựa trên sự hiện diện của thụ thể estrogen: thụ thể dương tính (ER+) hoặc thụ thể âm tính (ER−). Phytoestrogen calycosin đã được chứng minh là có khả năng ức chế sự phát triển của các tế bào ER+ có thể thông qua một vòng phản hồi liên quan đến miR-375, RAS dexamethasone-induced 1 (RASD1) và ERα. Tuy nhiên, cách mà calycosin tác động lên tế bào ung thư vú ER− vẫn chưa rõ ràng. Ở đây, chúng tôi chỉ ra rằng calycosin ức chế sự phát triển của cả tế bào ung thư vú ER− (MDA-MB-468 và SKBR3) và ER+ (MCF-7 và T47D) và các tác động ức chế này liên quan đến việc tăng cường biểu hiện của RNA không mã hóa dài (lncRNA) WDR7-7. Lần đầu tiên, chúng tôi chứng minh rằng biểu hiện của WDR7-7 giảm trong các dòng tế bào ung thư vú và việc quá biểu hiện WDR7-7 ức chế sự phát triển thông qua một cơ chế liên quan đến thụ thể estrogen liên kết với protein G 30 (GPR30). Đồng thời, chúng tôi cho thấy calycosin kích thích con đường tín hiệu WDR7-7-GPR30 trong các tế bào ung thư vú MCF-7, T47D, MDA-MB-468 và SKBR3. Ngược lại, trong các tế bào MCF10A và MDA-MB-231 thiếu GPR30, do thiếu WDR7-7-GPR30 để kích hoạt, calycosin không ức chế được sự phát triển tế bào. Ngoài ra, trong tất cả bốn dòng ung thư vú dương tính GPR30, calycosin làm giảm mức độ phosphoryl hóa của SRC, EGFR, ERK1/2 và Akt, nhưng việc ức chế WDR7-7 đã chặn những thay đổi này và tăng cường sự phát triển. Ở những con chuột mang mầm bệnh MCF-7 hoặc SKBR3, sự phát triển khối u bị ức chế bởi calycosin, và những thay đổi trong mức biểu hiện WDR7-7 và GPR30 trong mô khối u tương tự như trong các tế bào MCF-7 và SKBR3 nuôi cấy. Những kết quả này gợi ý rằng calycosin có thể ức chế sự phát triển của các tế bào ung thư vú, ít nhất một phần, thông qua tín hiệu WDR7-7-GPR30, điều này có thể giải thích lý do tại sao calycosin có thể tác động ức chế lên ung thư vú ER−.

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer with high proliferative activity. TNBC tumors exhibit elevated MYC expression and altered expression of MYC regulatory genes, which are associated with tumor progression and poor prognosis; however, the underlying mechanisms by which MYC retains its high expression and mediates TNBC tumorigenesis require further exploration. ACTL6A regulation of MYC and its target gene, CDK2, was defined using Co-IP, mass spectrometry and ChIP assays. To study the role of ACTL6A in TNBC, we performed soft-agar, colony formation, flow cytometry and tumor formation in nude mice. CDK2 inhibitor and paclitaxel were used in testing combination therapy in vitro and in vivo. ACTL6A bound MYC to suppress glycogen synthase kinase 3 beta (GSK3β)-induced phosphorylation on MYC T58, which inhibited ubiquitination of MYC and stabilized it. Moreover, ACTL6A promoted the recruitment of MYC and histone acetyltransferase KAT5 on CDK2 promoters, leading to hyperactivation of CDK2 transcription. ACTL6A overexpression promoted, while silencing ACTL6A suppressed cell proliferation and tumor growth in TNBC cells in vitro and in vivo, which was dependent on MYC signaling. Furthermore, co-therapy with paclitaxel and CDK2 inhibitor showed synergistic effects in tumor suppression. Notably, ACTL6A/MYC/CDK2 axis was specifically up-regulated in TNBC and high expression of ACTL6A was correlated to shorter survival in patients with TNBC. These findings reveal a novel mechanism by which ACTL6A prolongs the retention of MYC in TNBC and suggest that pharmacological targeting ACTL6A/MYC/CDK2 axis might have therapeutic potential in patients with TNBC.

We observed that paclitaxel altered the pharmacokinetic properties of gemcitabine in patients with non-small cell lung cancer (NSCLC) and limited the accumulation of gemcitabine and its metabolites in various primary and immortalized human cells. Therefore, we classified the drug-drug interaction and the effects of paclitaxel on deoxycytidine kinase (dCK) and cytidine deaminase (CDA) in three NSCLC cell lines. These enzymes are responsible for the metabolism of gemcitabine to its deaminated metabolite dFdU (80% of the parent drug) and the phosphorylated metabolites dFdCMP, dFdCDP and dFdCTP. These metabolites appear to relate to sensitivity and tolerability of gemcitabine based on previous animal and laboratory studies. Three immortalized human cells representative of the most common histological subtypes identified in patients with advanced NSCLC were exposed to the individual drugs or combinations to complete a multiple drug effect analysis. These same cell lines were exposed to vehicle-control or paclitaxel and the mRNA levels, protein expression and specific activity of dCK and CDA were compared. Comparisons were made using a two-tailed paired t-test or analysis of variance with a P value of < 0.05 considered significant. The multiple drug effect analysis indicated synergy for H460, H520 and H838 cells independent of sequence. As anticipated, paclitaxel-gemcitabine increased the number of G2/M cells, whereas gemcitabine-paclitaxel increased the number of G0/G1 or S cells. Paclitaxel significantly decreased dCK and CDA mRNA levels in H460 and H520 cells (40% to 60%, P < 0.05) and lowered dCK protein (24% to 56%, P < 0.05) without affecting CDA protein. However, paclitaxel increased both dCK (10% to 50%) and CDA (75% to 153%) activity (P < 0.05). Paclitaxel caused substantial declines in the accumulation of the deaminated and phosphorylated metabolites in H520 cells (P < 0.05); the metabolites were not measurable in the remaining two cell lines. The ratio of dCK to CDA mRNA levels corresponded to the combination index (CI) estimated for sequential paclitaxel-gemcitabine. In summary, paclitaxel altered the mRNA levels and specific activity of dCK and CDA and these effects could be dependent on histological subtype. More cell and animal studies are needed to further characterize the relationship between mRNA levels and the overall drug-drug interaction and the potential to use histological subtype as a predictive factor in the selection of an appropriate anticancer drug regimen.

Hedgehog-Gli1 signaling induces development of two common neurological features seen in pancreatic ductal adenocarcinoma (PDAC): peripheral neural invasion (PNI) and peripheral neural remodeling (PNR). However, the underlying molecular mechanisms in cancer cells and nerves within Gli1-derived PNR have not previously been comprehensively analyzed. In this study, RNA sequencing was used to screen meaningful circRNAs in PNR. An in vitro model of PNR was subsequently constructed through a co-culture system comprising PDAC cells and murine dorsal root ganglia (DRG) (as the neuronal element), and the relevant mechanisms were explored using a series of molecular biology experiments. A subcutaneous nude mouse tumorigenesis model was established to further verify the occurrence of PNR that was detected in human PDAC samples. We first confirmed the molecular mechanisms of PNR development through crosstalk between exosomal circ-0011536 and DRG. In Gli1-overpressed PDAC, circ-0011536 is mainly secreted by exosomes. After being ingested by DRG, it can promote the activity of DRG by degrading miR-451a and upregulating the expression of VGF. Overexpression of Gli1 can accelerate the proliferation of subcutaneous tumors in mice and is closely related to the density of nerve plexuses, while downregulating circ-RNA inhibits tumor proliferation and reduces the density of nerve plexuses. In addition, TMA results confirmed that Gli1 overexpression significantly increased the expression of VGF and was closely associated with increased nerve plexus density. Hedgehog-Gli1-induced exosomal circ-0011536 promoted PNR via the miR-451a/VGF axis, thereby establishing that it may contribute to PDAC-associated nerve changes with activated Hedgehog signaling.

Pancreatic cancer is one of the most difficult cancers to detect early and most patients die from complications arising due to distant organ metastases. The lack of bona fide early biomarkers is one of the primary reasons for late diagnosis of pancreatic cancer. It is a multifactorial disease and warrants a novel approach to identify early biomarkers. In order to characterize the proteome, Extracellular vesicles (EVs) isolated from different in vitro conditions mimicking tumor-microenvironment interactions between pancreatic cancer epithelial and stromal cells were analyzed using high throughput mass spectrometry. The biological activity of the secreted EVome was analyzed by investigating changes in distant organ metastases and associated early changes in the microbiome. Candidate biomarkers (KIF5B, SFRP2, LOXL2, and MMP3) were selected and validated on a mouse-human hybrid Tissue Microarray (TMA) that was specifically generated for this study. Additionally, a human TMA was used to analyze the expression of KIF5B and SFRP2 in progressive stages of pancreatic cancer. The EVome of co-cultured epithelial and stromal cells is different from individual cells with distinct protein compositions. EVs secreted from stromal and cancer cells cultures could not induce significant changes in Pre-Metastatic Niche (PMN) modulation, which was assessed by changes in the distant organ metastases. However, they did induce significant changes in the early microbiome, as indicated by differences in α and β-diversities. KIF5B and SFRP2 show promise for early detection and investigation in progressive pancreatic cancer. These markers are expressed in all stages of pancreatic cancer such as low grade PanINs, advanced cancer, and in liver and soft tissue metastases. Proteomic characterization of EVs derived from mimicking conditions of epithelial and stromal cells in the tumor-microenvironment resulted in the identification of several proteins, some for the first time in EVs. These secreted EVs cannot induce changes in distant organ metastases in in vivo models of EV education, but modulate changes in the early murine microbiome. Among all the proteins that were analyzed (MMP3, KIF5B, SFRP2, and LOXL2), KIF5B and SFRP2 show promise as bona fide early pancreatic cancer biomarkers expressed in progressive stages of pancreatic cancer.

In order to develop a new immunotherapeutic agent targeting metastatic breast cancers, we chose to utilize autocatalytic feature of the membrane serine protease Prss14/ST14, a specific prognosis marker for ER negative breast cancer as a target molecule. The study was conducted using three mouse breast cancer models, 4 T1 and E0771 mouse breast cancer cells into their syngeneic hosts, and an MMTV-PyMT transgenic mouse strain was used. Prss14/ST14 knockdown cells were used to test function in tumor growth and metastasis, peptides derived from the autocatalytic loop for activation were tested as preventive metastasis vaccine, and monoclonal and humanized antibodies to the same epitope were tested as new therapeutic candidates. ELISA, immunoprecipitation, Immunofluorescent staining, and flow cytometry were used to examine antigen binding. The functions of antibodies were tested in vitro for cell migration and in vivo for tumor growth and metastasis. Prss14/ST14 is critically involved in the metastasis of breast cancer and poor survival rather than primary tumor growth in two mouse models. The epitopes derived from the specific autocatalytic loop region of Prss14/ST14, based on structural modeling acted as efficient preventive metastasis vaccines in mice. A new specific monoclonal antibody mAb3F3 generated against the engineered loop structure could reduce cell migration, eliminate metastasis in PyMT mice, and can detect the Prss14/ST14 protein expressed in various human cancer cells. Humanized antibody huAb3F3 maintained the specificity and reduced the migration of human breast cancer cells in vitro. Our study demonstrates that Prss14/ST14 is an important target for modulating metastasis. Our newly developed hybridoma mAbs and humanized antibody can be further developed as new promising candidates for the use in diagnosis and in immunotherapy of human metastatic breast cancer.

Immunotherapy has emerged as a promising strategy for boosting antitumoral immunity. Blockade of immune checkpoints (ICs), which regulate the activity of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells has proven clinical benefits. Antibodies targeting CTLA-4, PD-1, and PD-L1 are IC-blockade drugs approved for the treatment of various solid and hematological malignancies. However, a large subset of patients does not respond to current anti-IC immunotherapy. An integrative understanding of tumor-immune infiltrate, and IC expression and function in immune cell populations is fundamental to the design of effective therapies. The simultaneous blockade of newly identified ICs, as well as of previously described ICs, could improve antitumor response. We review the potential for novel combinatory blockade strategies as antitumoral therapy, and their effects on immune cells expressing the targeted ICs. Preclinical evidence and clinical trials involving the blockade of the various ICs are reported. We finally discuss the rationale of IC co-blockade strategy with respect to its downstream signaling in order to improve effective antitumoral immunity and prevent an increased risk of immune-related adverse events (irAEs).