Inhibition of NEK7 Suppressed Hepatocellular Carcinoma Progression by Mediating Cancer Cell Pyroptosis.

NIMA-related kinase 7 (NEK7) is a serine/threonine kinase involved in cell cycle progression via mitotic spindle formation and cytokinesis. It has been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer, and colorectal cancer. Moreover, NEK7 regulated the NLRP3 inflammasome to activate Caspase-1, resulting in cell pyroptosis. In the present study, we investigated whether NEK7 is involved in cell pyroptosis of hepatocellular carcinoma (HCC). Interestingly, we found that NEK7 was significantly related to expression of pyroptosis marker GSDMD in HCC. We found that NEK7 expression was significantly correlated with GSDMD expression in bioinformatics analysis, and NEK7 expression was significantly co-expressed with GSDMD in our HCC specimens. Cell viability, migration, and invasion capacity of HCC cell lines were inhibited, and the tumor growth in the xenograft mouse model was also suppressed following knockdown of NEK7 expression. Mechanistic studies revealed that knockdown of NEK7 in HCC cells significantly upregulated the expression of pyroptosis markers such as NLRP3, Caspase-1, and GSDMD. Coculture of HCC cells stimulated hepatic stellate cell activation by increasing p-ERK1/2 and α-SMA. Knockdown of NEK7 impaired the stimulation of HCC cells. Therefore, downregulation of NEK7 inhibited cancer-stromal interaction by triggering cancer cell pyroptosis. Taken together, this study highlights the functional role of NEK7-regulated pyroptosis in tumor progression and cancer-stromal interaction of HCC, suggesting NEK7 as a potential target for a new therapeutic strategy of HCC treatment.

Novel targets identified by integrated cancer-stromal interactome analysis of pancreatic adenocarcinoma.

The pancreatic cancer microenvironment is crucial in cancer development, progression and drug resistance. Cancer-stromal interactions have been recognized as important targets for cancer therapy. However, identifying relevant and druggable cancer-stromal interactions is challenging due to the lack of quantitative methods to analyze the whole cancer-stromal interactome. Here we studied 14 resected pancreatic cancer specimens (8 pancreatic adenocarcinoma (PDAC) patients as a cancer group and 6 intraductal papillary-mucinous adenoma (IPMA) patients as a control). Shotgun proteomics of the stromal lesion dissected with laser captured microdissection (LCM) was performed, and identified 102 differentially expressed proteins in pancreatic cancer stroma. Next, we obtained gene expression data in human pancreatic cancer and normal pancreatic tissue from The Cancer Genome Atlas database (n = 169) and The Genotype-Tissue Expression database (n = 197), and identified 1435 genes, which were differentially expressed in pancreatic cancer cells. To identify relevant and druggable cancer-stromal-interaction targets, we applied these datasets to our in-house ligand-receptor database. Finally, we identified 9 key genes and 8 key cancer-stromal-interaction targets for PDAC patients. Furthermore, we examined FN1 and ITGA3 protein expression in pancreatic cancer tissues using tissue microarrays (TMAs) of 271 PDAC cases, and demonstrated that FN1-ITGA3 had unfavorable prognostic impact for PDAC patients.

Cancer-stromal cell fusion as revealed by fluorescence protein tracking.

PURPOSE: We previously determined that cancer-stromal interaction was a direct route to tumor cell heterogeneity progression, since cancer-stromal cell fusion in coculture resulted in the creation of heterogeneous clones of fusion hybrid progeny. In this report, we modified the cancer-stromal coculture system to establish optimal experimental conditions for investigating cell fusion machinery and the mechanism of heterogeneity progression. EXPERIMENTAL DESIGN: Red fluorescence protein-tagged LNCaP cells were cocultured with green fluorescence protein-labeled prostate stromal cells for cancer-stromal cell fusion, which was tracked as dual fluorescent cells by fluorescence microscopy. RESULTS: We identified the most efficient strategy to isolate clones of fusion hybrid progenies. From the coculture, mixed cells including fusion hybrids were subjected to low-density replating for colony formation by fusion hybrid progeny. These colonies could propagate into derivative cell populations. Compared to the parental LNCaP cells, clones of the fusion hybrid progeny displayed divergent behaviors and exhibited permanent genomic hybridization. CONCLUSIONS: Cancer-stromal cell fusion leads to cancer cell heterogeneity. The cancer-stromal coculture system characterized in this study can be used as a model for molecular characterization of cancer cell fusion as the mechanism behind the progression of heterogeneity observed in clinical prostate cancers.

Inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer-stromal interaction and metastasis.

BACKGROUND: Extracellular signal-regulated kinases (ERKs) have been related to multiple cancers, including breast cancer, hepatocellular cancer, lung cancer and colorectal cancer. ERK1/2 inhibitor can suppress growth of KRAS-mutant pancreatic tumors by targeting cancer cell. However, no studies have shown the expression of ERK1/2 on pancreatic stromal and its effect on pancreatic cancer-stromal interaction. METHODS: Immunohistochemistry and western blotting were performed to detect the expression of p-ERK1/2 in pancreatic tissues and cells. Cell viability assay was used to study IC50 of ERK inhibitor on pancreatic cancer cells (PCCs) and primary cancer-associated pancreatic stellate cells (PSCs). Transwell migration, invasion, cell viability assay, senescence ß-galactosidase staining were performed to determine the effect of ERK inhibitor on PCCs and PSCs in vitro and in vivo. The expression of key factors involved in autophagy and epithelial-to-mesenchymal transition (EMT) process were evaluated by western blotting. The expression of key factors related to cell invasiveness and malignancy were confirmed by qRT-PCR. Co-transplantation of PCC Organoid and PSC using a splenic xenograft mouse model was used to evaluated combined treatment of ERK inhibitor and autophagy inhibitor. RESULTS: Immunohistochemical staining in pancreatic tumor samples and transgenetic mice detected p-ERK1/2 expression in both cancer cells and stromal cells. In pancreatic tissues, p-ERK1/2 was strongly expressed in cancer-associated PSCs compared with cancer cells and normal PSCs. PSCs were also significantly more sensitive to ERK1/2 inhibitor treatment. Inhibition of ERK1/2 suppressed EMT transition in HMPCCs, upregulated cellular senescence markers, activated autophagy in cancer-associated PSCs; and suppressed cancer-stromal interaction, which enhanced invasiveness and viability of cancer cells. We also found that chloroquine, an autophagy inhibitor, suppressed ERK inhibition-induced autophagy and promoted PSC cellular senescence, leading to significantly decreased cell proliferation. The combination of an ERK inhibitor and autophagy inhibitor suppressed liver metastasis in a splenic pancreatic cancer organoid xenograft mouse model. CONCLUSIONS: These data indicate that inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer-stromal interaction and metastasis.

Regulatory signaling network in the tumor microenvironment of prostate cancer bone and visceral organ metastases and the development of novel therapeutics.

This article describes cell signaling network of metastatic prostate cancer (PCa) to bone and visceral organs in the context of tumor microenvironment and for the development of novel therapeutics. The article focuses on our recent progress in the understanding of: 1) The plasticity and dynamics of tumor-stroma interaction; 2) The significance of epigenetic reprogramming in conferring cancer growth, invasion and metastasis; 3) New insights on altered junctional communication affecting PCa bone and brain metastases; 4) Novel strategies to overcome therapeutic resistance to hormonal antagonists and chemotherapy; 5) Genetic-based therapy to co-target tumor and bone stroma; 6) PCa-bone-immune cell interaction and TBX2-WNTprotein signaling in bone metastasis; 7) The roles of monoamine oxidase and reactive oxygen species in PCa growth and bone metastasis; and 8) Characterization of imprinting cluster of microRNA, in tumor-stroma interaction. This article provides new approaches and insights of PCa metastases with emphasis on basic science and potential for clinical translation. This article referenced the details of the various approaches and discoveries described herein in peer-reviewed publications. We dedicate this article in our fond memory of Dr. Donald S. Coffey who taught us the spirit of sharing and the importance of focusing basic science discoveries toward translational medicine.

Low-dose eribulin mesylate exerts antitumor effects in gastric cancer by inhibiting fibrosis via the suppression of epithelial-mesenchymal transition and acts synergistically with 5-fluorouracil.

BACKGROUND: Characterized by aggressive proliferation, extensive stromal fibrosis, and resulting drug resistance, peritoneal dissemination in gastric cancer remains associated with poor prognosis. Interaction between cancer and stromal cells accelerates tumor progression via epithelial-mesenchymal transition (EMT), which is one of the major causes of tissue fibrosis, and human peritoneal mesothelial cells (HPMCs) play important roles as cancer stroma in peritoneal dissemination. Transforming growth factor-ß (TGF-ß) has a pivotal function in the progression of EMT, and Smad proteins play an important role in the TGF-ß signaling pathway. Eribulin mesylate (eribulin), a nontaxane microtubule dynamics inhibitor used for the treatment of advanced breast cancer, inhibits EMT changes in triple-negative breast cancer cells. We examined its ability to inhibit tumor progression and EMT changes resulting from the interaction between gastric cancer cells and HPMCs and to act synergistically with 5-fluorouracil (5-FU), a key drug for gastric cancer. MATERIALS AND METHODS: Proliferation of gastric cancer cells and HPMCs isolated from healthy omentum was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Following gastric cancer cell/HPMC coculture, EMT markers were detected by immunofluorescence, immunohistochemistry, and Western blotting; invasion assays were performed; and TGF-ß and Smad phosphorylation were assessed by Western blotting and enzyme-linked immunosorbent assay. A mouse fibrotic tumor xenograft model was established using gastric cancer cell/HPMC cocultures. The effect of eribulin and/or 5-FU was tested in each case. RESULTS: Eribulin significantly suppressed gastric cancer cell proliferation and EMT changes in MKN-45 gastric cancer cells and HPMCs induced by their interaction in vitro. Eribulin inhibited EMT at much lower concentrations (≥0.5 nM for MKN-45 and ≥0.1 nM for HPMCs) than its half maximal inhibitory concentrations (2.2 nM for MKN-45 and 8.1 nM for HPMCs), and this resulted, at least partly, from the downregulation of TGF-ß/Smad signaling. Eribulin administration of ≥0.1 mg/kg suppressed tumor progression (0.1 mg/kg, p=0.02), and fibrosis was inhibited by lower dose (0.05 mg/kg, p=0.008) in the xenograft model. Furthermore, 0.05 mg/kg administration with 5-FU brought about synergistic antitumor effects (p=0.006). CONCLUSION: Low-dose eribulin combined with 5-FU might be a promising therapy for peritoneal dissemination in gastric cancer.

Calpain inhibitor calpeptin suppresses pancreatic cancer by disrupting cancer-stromal interactions in a mouse xenograft model.

Desmoplasia contributes to the aggressive behavior of pancreatic cancer. However, recent clinical trials testing several antifibrotic agents on pancreatic cancer have not shown clear efficacy. Therefore, further investigation of desmoplasia-targeting antifibrotic agents by another mechanism is needed. Calpeptin, an inhibitor of calpains, suppressed fibroblast function and inhibited fibrosis. In this study, we investigated the anticancer effects of calpeptin on pancreatic cancer. We investigated whether calpeptin inhibited tumor progression using a mouse xenograft model. We used quantitative RT-PCR to evaluate the expression of calpain-1 and calpain-2 mRNA in pancreatic cancer cells (PCCs) and pancreatic stellate cells (PSCs). We also undertook functional assays, including proliferation, migration, and invasion, to evaluate the inhibitory effects of calpeptin on PCCs and PSCs. Quantitative RT-PCR indicated that PCCs and PSCs expressed calpain-2 mRNA. Calpeptin reduced tumor volume (P = 0.0473) and tumor weight (P = 0.0471) and inhibited the tumor desmoplastic reaction (P < 0.001) in xenograft tumors in nude mice. Calpeptin also inhibited the biologic functions of PCCs and PSCs including proliferation (P = 0.017), migration (P = 0.027), and invasion (P = 0.035) in vitro. Furthermore, calpeptin reduced the migration of PCCs and PSCs by disrupting the cancer-stromal interaction (P = 0.0002). Our findings indicate that calpeptin is a promising antitumor agent for pancreatic cancer, due not only to its suppressive effect on PCCs and PSCs but also its disruption of the cancer-stromal interaction.

Differential effects of adipose tissue stromal cells on the apoptosis, growth and invasion of bladder urothelial carcinoma between the superficial and invasive types.

OBJECTIVES: To clarify the interaction between adipose tissue stromal cells and bladder cancer cells. METHODS: Superficial (RT4) and invasive (EJ) urothelial carcinoma cells were cultured on adipose tissue stromal cell-embedded or non-embedded collagen gel. Cells were analyzed by immunohistochemistry, western blot and real-time reverse transcription polymerase chain reaction. RESULTS: Adipose tissue stromal cells inhibited growth of RT4, while they promoted the apoptosis. In contrast, adipose tissue stromal cells promoted growth of EJ, but they did not affect the apoptosis. Adipose tissue stromal cells slightly promoted expression of mitogen-activated protein kinase cascade in RT4 and EJ. Adipose tissue stromal cells promoted display of the molecular-targeted agent human epidermal growth factor receptor-2 in only RT4. In turn, RT4 and EJ enhanced α-smooth muscle actin (myofibroblast marker) and S-100 protein (adipocyte marker) expression of adipose tissue stromal cells, respectively. CONCLUSIONS: These findings suggest that: (i) adipose tissue stromal cells might suppress the progression of superficial-type cancer, whereas they might promote that of invasive type; (ii) adipose tissue stromal cell-activated mitogen-activated protein kinase pathway might play differential roles in both types of bladder cancer; (iii) human epidermal growth factor receptor-2 could represent a critical therapeutic agent for the superficial type under adipose tissue stromal cells-cancer interaction; and (iv) superficial bladder cancer might promote myofibroblast differentiation of adipose tissue stromal cells as a cancer-associate phenotype, whereas invasive bladder cancer might promote their adipocyte differentiation.