TORC1 and class I HDAC inhibitors synergize to suppress mature B cell neoplasms

Enhanced proliferative signaling and loss of cell cycle regulation are essential for cancer progression. Increased mitogenic signaling through activation of the mTOR pathway, coupled with deregulation of the Cyclin D/retinoblastoma (Rb) pathway is a common feature of lymphoid malignancies, including plasmacytoma (PCT), multiple myeloma (MM), Burkitt''s lymphoma (BL), and mantle cell lymphoma (MCL). Here we evaluate the synergy of pharmacologically affecting both of these critical pathways using the mTOR inhibitor sirolimus and the histone deacetylase inhibitor entinostat. A dose‐matrix screening approach found this combination to be highly active and synergistic in a panel of genetically diverse human MM cell lines. Synergy and activity was observed in mouse PCT and human BL and MCL cell lines tested in vitro, as well as in freshly isolated primary MM patient samples tested ex vivo. This combination had minimal effects on healthy donor cells and retained activity when tested in a co‐culture system simulating the protective interaction of cancer cells with the tumor microenvironment. Combining sirolimus with entinostat enhanced cell cycle arrest and apoptosis. At the molecular level, entinostat increased the expression of cell cycle negative regulators including CDKN1A (p21) and CDKN2A (p16), while the combination decreased critical growth and survival effectors including Cyclin D, BCL‐XL, BIRC5, and activated MAPK.     

Nuclear accumulation of KPNA2 impacts radioresistance through positive regulation of the PLSCR1‐STAT1 loop in lung adenocarcinoma

Lung adenocarcinoma (ADC) is the predominant histological type of lung cancer, and radiotherapy is one of the current therapeutic strategies for lung cancer treatment. Unfortunately, biological complexity and cancer heterogeneity contribute to radioresistance development. Karyopherin α2 (KPNA2) is a member of the importin α family that mediates the nucleocytoplasmic transport of cargo proteins. KPNA2 overexpression is observed across cancer tissues of diverse origins. However, the role of KPNA2 in lung cancer radioresistance is unclear. Herein, we demonstrated that high expression of KPNA2 is positively correlated with radioresistance and cancer stem cell (CSC) properties in lung ADC cells. Radioresistant cells exhibited nuclear accumulation of KPNA2 and its cargos (OCT4 and c‐MYC). Additionally, KPNA2 knockdown regulated CSC‐related gene expression in radioresistant cells. Next‐generation sequencing and bioinformatic analysis revealed that STAT1 activation and nuclear phospholipid scramblase 1 (PLSCR1) are involved in KPNA2‐mediated radioresistance. Endogenous PLSCR1 interacting with KPNA2 and PLSCR1 knockdown suppressed the radioresistance induced by KPNA2 expression. Both STAT1 and PLSCR1 were found to be positively correlated with dysregulated KPNA2 in radioresistant cells and ADC tissues. We further demonstrated a potential positive feedback loop between PLSCR1 and STAT1 in radioresistant cells, and this PLSCR1‐STAT1 loop modulates CSC characteristics. In addition, AKT1 knockdown attenuated the nuclear accumulation of KPNA2 in radioresistant lung cancer cells. Our results collectively support a mechanistic understanding of a novel role for KPNA2 in promoting radioresistance in lung ADC cells.     

金属硫蛋白1h与常染色质组蛋白甲基转移酶1结合调节组蛋白H3K9甲基化并抑制肺癌细胞周期及运动

目的:观察常染色质组蛋白甲基转移酶1(euchromatic histone methyltransferase1,EHMT1)与金属硫蛋白1h(metallothionein1h,MT1h)的相互作用及定位,探讨其对组蛋白H3K9甲基化和肺癌细胞生物学行为的影响及可能的作用机制。方法:将pGBKT7-MT1h质粒与pGADT7-EHMT1质粒共同转化至酵母菌株AH109,进行酵母双杂交实验。将pcDNA4/TO/myc-hisA/MT1h与pcDNA6/TR质粒共转染肺癌细胞株A549和A2,并用四环素诱导MT1h表达后,采用免疫共沉淀和激光共聚焦法检测MT1h、标签蛋白c-myc和EHMT1蛋白的表达及定位;运用组蛋白H3K9甲基转移酶试剂盒检测甲基化转移酶活性,Western印迹法检测三甲基化的组蛋白H3K9表达;FCM和克隆形成实验检测MT1h表达对细胞增殖的影响;划痕实验和Borden小室法检测MT1h对细胞迁移的影响。结果:实验证实MT1h与EHMT1存在直接作用,且二者共定位于细胞核。诱导MT1h表达后,组蛋白甲基化转移酶活性升高,并且MT1h与组蛋白赖氨酸H3K9三甲基化密切相关。功能分析显示,MT1h稳定转染的肺癌细胞多处于G0期,细胞运动和迁移能力降低。结论:MT1h与EHMT1在细胞核内直接作用,可能通过调节组蛋白H3K9甲基化来发挥抑癌基因的作用。     

SAR1A regulates the RhoA/YAP and autophagy signaling pathways to influence osteosarcoma invasion and metastasis

Osteosarcoma is the most prevalent form of primary bone malignancy affecting adolescents. Secretion‐associated Ras‐related GTPase 1A (SAR1A) is a key regulator of endoplasmic reticulum (ER) homeostasis, but its role as a regulator of osteosarcoma metastasis has yet to be clarified. Bioinformatics analyses revealed SAR1A and RHOA to be upregulated in osteosarcoma patients, with the upregulation of these genes being associated with poor 5‐year metastasis‐free survival rates. In addition, the upregulation of SAR1A and RHOA in osteosarcoma was highly positively correlated. Immunohistochemical analyses additionally revealed that SAR1A levels were increased in osteosarcoma pulmonary metastases. In vitro wound healing and Transwell assays indicated that knocking down SAR1A or RHOA impaired the invasive and migratory activity of osteosarcoma cells, whereas RHOA overexpression had the opposite effect. Western blotting and immunofluorescent staining revealed the inhibition of osteosarcoma cell epithelial–mesenchymal transition following SAR1A or RHOA knockdown; RHOA overexpression had the opposite effect. Following SAR1A knockdown, phalloidin staining indicated that osteosarcoma cells showed reduced lamellipodia formation. Endoplasmic reticulum stress levels and reactive oxygen species production were enhanced following the knockdown of SAR1A, as was autophagic activity, with lung metastases being reduced in vivo after such knockdown. Knocking down SAR1A suppresses osteosarcoma cell metastasis through the RhoA/YAP, ER stress, and autophagic pathways, offering new insights into the regulation of autophagic activity in the context of osteosarcoma cell metastasis and suggesting that these pathways could be amenable to therapeutic intervention.     

ARID1A‐deficient cells require HDAC6 for progression of endometrial carcinoma

AT‐rich interactive domain‐containing protein 1A (ARID1A) loss‐of‐function mutation accompanied by a loss of ARID1A protein expression is frequently observed in endometrial carcinomas. However, the molecular mechanisms linking these genetic changes to the altered pathways regulating tumour initiation, maintenance and/or progression remain poorly understood. Thus, the main aim of this study was to analyse the role of ARID1A loss of function in endometrial tumorigenesis. Here, using different endometrial in vitro and in vivo models, such as tumoral cell lines, 3D primary cultures and metastatic or genetically modified mouse models, we show that altered expression of ARID1A is not enough to initiate endometrial tumorigenesis. However, in an established endometrial cancer context, ARID1A loss of function accelerates tumoral progression and metastasis through the disruption of the G2/M cell cycle checkpoint and ATM/ATR‐mediated DNA damage checkpoints, increases epithelial cell proliferation rates and induces epithelial mesenchymal transition through the activation of histone deacetylase 6 (HDAC6). Next, we demonstrated that the inhibition of HDAC6 function, using the HDAC6‐specific inhibitor ACY1215 or by transfection with HDAC6 short hairpin RNA (shRNA), can reverse the migratory and invasive phenotype of ARID1A‐knockdown cells. Further, we also show that inhibition of HDAC6 activity causes an apoptotic vulnerability to etoposide treatments in ARID1A‐deficient cells. In summary, the findings exposed in this work indicate that the inhibition of HDAC6 activity is a potential therapeutic strategy for patients suffering from ARID1A‐mutant endometrial cancer diagnosed in advanced stages.     

Acquired small cell lung cancer resistance to Chk1 inhibitors involves Wee1 up‐regulation

Platinum‐based chemotherapy has been the cornerstone treatment for small cell lung cancer (SCLC) for decades, but no major progress has been made in the past 20 years with regard to overcoming chemoresistance. As the cell cycle checkpoint kinase 1 (Chk1) plays a key role in DNA damage response to chemotherapeutic drugs, we explored the mechanisms of acquired drug resistance to the Chk1 inhibitor prexasertib in SCLC. We established prexasertib resistance in two SCLC cell lines and found that DNA copy number, messengerRNA (mRNA) and protein levels of the cell cycle regulator Wee1 significantly correlate with the level of acquired resistance. Wee1 small interfering RNA (siRNA) or Wee1 inhibitor reversed prexasertib resistance, whereas Wee1 transfection induced prexasertib resistance in parental cells. Reverse phase protein microarray identified up‐regulated proteins in the resistant cell lines that are involved in apoptosis, cell proliferation and cell cycle. Down‐regulation of CDK1 and CDC25C kinases promoted acquired resistance in parental cells, whereas down‐regulation of p38MAPK reversed the resistance. High Wee1 expression was significantly correlated with better prognosis of resected SCLC patients. Our results indicate that Wee1 overexpression plays an important role in acquired resistance to Chk1 inhibition. We also show that bypass activation of the p38MAPK signaling pathway may contribute to acquired resistance to Chk1 inhibition. The combination of Chk1 and Wee1 inhibitors may provide a new therapeutic strategy for the treatment of SCLC.     

Pivotal role of epithelial cell adhesion molecule in the survival of lung cancer cells

Epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of human cancers including lung cancer, and its contribution to increased proliferation through upregulation of cell cycle accelerators such as cyclins A and E has been well established in breast and gastric cancers. Nevertheless, very little is known about its role in supporting the survival of cancer cells. In addition, the functional role of EpCAM in the pathogenesis of lung cancer remains to be explored. In this study, we show that RNAi-mediated knockdown of EpCAM suppresses proliferation and clonogenic growth of three EpCAM-expressing lung cancer cell lines (H3255, H358, and HCC827), but does not induce cell cycle arrest in any of these. In addition, EpCAM knockdown inhibits invasion in the highly invasive H358 but not in less invasive H3255 cells in a Transwell assay. Of note, the EpCAM knockdown induces massive apoptosis in the three cell lines as well as in another EpCAM-expressing lung cancer cell line, HCC2279, but to a much lesser extent in a cdk4/hTERT immortalized normal human bronchial epithelial cell line, HBEC4, suggesting that EpCAM could be a therapeutic target for lung cancer. Finally, EpCAM knockdown partially restores contact inhibition in HCC827, in association with p27(Kip1) upregulation. These results indicate that EpCAM could contribute substantially to the pathogenesis of lung cancer, especially cancer cell survival, and suggest that EpCAM targeted therapy for lung cancer may have potential.     

PHOSPHATE exporter XPR1/SLC53A1 is required for the tumorigenicity of epithelial ovarian cancer

Ovarian cancer is the fifth most common cause of cancer‐related death in women. Ovarian clear cell carcinoma (OCCC) is a chemotherapy‐resistant epithelial ovarian cancer with poor prognosis. As a basis for the development of therapeutic agents that could improve the prognosis of OCCC, we performed a screen for proteins critical for the tumorigenicity of OCCC using the CRISPR/Cas9 system. Here we show that knockdown of the phosphate exporter XPR1/SLC53A1 induces the growth arrest and apoptosis of OCCC cells in vitro. Moreover, we show that knockdown of XPR1/SLC53A1 inhibits the proliferation of OCCC cells xenografted into immunocompromised mice. These results suggest that XPR1/SLC53A1 plays a critical role in the tumorigenesis of OCCC cells. We speculate that XPR1/SLC53A1 might be a promising molecular target for the therapeutic treatment of OCCC.     

The SKP2‐p27 axis defines susceptibility to cell death upon CHK1 inhibition

Checkpoint kinase 1 (CHK1; encoded by CHEK1) is an essential gene that monitors DNA replication fidelity and prevents mitotic entry in the presence of under‐replicated DNA or exogenous DNA damage. Cancer cells deficient in p53 tumor suppressor function reportedly develop a strong dependency on CHK1 for proper cell cycle progression and maintenance of genome integrity, sparking interest in developing kinase inhibitors. Pharmacological inhibition of CHK1 triggers B‐Cell CLL/Lymphoma 2 (BCL2)‐regulated cell death in malignant cells largely independently of p53, and has been suggested to kill p53‐deficient cancer cells even more effectively. Next to p53 status, our knowledge about factors predicting cancer cell responsiveness to CHK1 inhibitors is limited. Here, we conducted a genome‐wide CRISPR/Cas9‐based loss‐of‐function screen to identify genes defining sensitivity to chemical CHK1 inhibitors. Next to the proapoptotic BCL2 family member, BCL2 Binding Component 3 (BBC3; also known as PUMA), the F‐box protein S‐phase Kinase‐Associated Protein 2 (SKP2) was validated to tune the cellular response to CHK1 inhibition. SKP2 is best known for degradation of the Cyclin‐dependent Kinase Inhibitor 1B (CDKN1B; also known as p27), thereby promoting G1‐S transition and cell cycle progression in response to mitogens. Loss of SKP2 resulted in the predicted increase in p27 protein levels, coinciding with reduced DNA damage upon CHK1‐inhibitor treatment and reduced cell death in S‐phase. Conversely, overexpression of SKP2, which consequently results in reduced p27 protein levels, enhanced cell death susceptibility to CHK1 inhibition. We propose that assessing SKP2 and p27 expression levels in human malignancies will help to predict the responsiveness to CHK1‐inhibitor treatment.     

A small molecule targeting CHI3L1 inhibits lung metastasis by blocking IL‐13Rα2‐mediated JNK‐AP‐1 signals

Our previous big data analyses showed a high level of association between chitinase 3 like1 (CHI3L1) expression and lung tumor development. In the present study, we investigated whether a CHI3L1‐inhibiting chemical, 2‐({3‐[2‐(1‐cyclohexen‐1‐yl)ethyl]‐6,7‐dimethoxy‐4‐oxo‐3,4‐dihydro‐2‐quinazolinyl}sulfanyl)‐N‐(4‐ethylphenyl)butanamide (K284), could inhibit lung metastasis and studied its mechanism of action. We investigated the antitumor effect of K284 both in vitro and in vivo. K284 (0.5 mg·kg⁻¹ body weight) significantly inhibited lung metastasis in in vivo models after injection of murine melanoma cells (B16F10) or adenocarcinomic human alveolar basal epithelial cells (A549). K284 significantly and concentration‐dependently also inhibited cancer cell proliferation and migration in the A549 and H460 lung cancer cell lines. We found that the binding of K284 to the chitin‐binding domain (CBD) of CHI3L1 prevented the binding of CHI3L1 to its receptor, interleukin‐13 receptor subunit alpha‐2 (IL‐13Rα2), thereby suppressing the CHI3L1 signal. This blocking of the CHI3L1‐IL‐13Rα2 signal caused the inhibition of c‐Jun N‐terminal kinase (JNK)‐activator protein 1 (AP‐1) signals, resulting in the prevention of lung metastasis and cancer cell growth. Our data demonstrate that K284 may serve as a potential candidate anticancer compound targeting CHI3L1.     

Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer

Purpose

Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells.

Methods

A lentivirus-mediated short hairpin RNA (shRNA) was designed to knockdown the expression of PP4R1 in ZR-75-30 breast cancer cells. The efficiency of lentivirus-mediated shRNA infection was determined using fluorescence microscopy to observe lentivirus-mediated green fluorescent protein expression and confirmed to be over 80%. PP4R1 expression in infected ZR-75-30 cells was detected by quantitative real-time polymerase chain reaction and western blot analysis. Cell proliferation and colony formation ability were measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and colony formation assay, respectively. Flow cytometry was used to measure cell cycle progression and cell apoptosis. In addition, apoptosis makers, including poly-ADP-ribose polymerase (PARP) and caspase-3, were investigated in PP4R1-silenced ZR-75-30 cells by western blot assay.

Results

We successfully constructed lentivirus-mediated shRNA to target PP4R1 in ZR-75-30 cells. MTT assay and colony formation assay showed the loss of PP4R1 suppressed the proliferation of ZR-75-30 cells. Flow cytometry analysis indicated cell cycle arrest and increased cell apoptosis in PP4R1 knockdown cells. Further, the apoptosis response in cells depleted of PP4R1 was illustrated by downregulation of PARP and upregulation of caspase-3.

Conclusion

Our results suggest that PP4R1 could promote breast cancer cell proliferation and might play a vital role in breast cancer occurrence.     

Activated macrophages promote invasion by early colorectal cancer via an interleukin 1β‐serum amyloid A1 axis

Submucosal invasion and lymph node metastasis are important issues affecting treatment options for early colorectal cancer (CRC). In this study, we aimed to unravel the molecular mechanism underlying the invasiveness of early CRCs. We performed RNA‐sequencing (RNA‐seq) with poorly differentiated components (PORs) and their normal counterparts isolated from T1 CRC tissues and detected significant upregulation of serum amyloid A1 (SAA1) in PORs. Immunohistochemical analysis revealed that SAA1 was specifically expressed in PORs at the invasive front of T1b CRCs. Upregulation of SAA1 in CRC cells promoted cell migration and invasion. Coculture experiments using CRC cell lines and THP‐1 cells suggested that interleukin 1β (IL‐1β) produced by macrophages induces SAA1 expression in CRC cells. Induction of SAA1 and promotion of CRC cell migration and invasion by macrophages were inhibited by blocking IL‐1β. These findings were supported by immunohistochemical analysis of primary T1 CRCs showing accumulation of M1‐like/M2‐like macrophages at SAA1‐positive invasive front regions. Moreover, SAA1 produced by CRC cells stimulated upregulation of matrix metalloproteinase‐9 in macrophages. Our data suggest that tumor‐associated macrophages at the invasive front of early CRCs promote cancer cell migration and invasion through induction of SAA1 and that SAA1 may be a predictive biomarker and a useful therapeutic target.