RASSF1A independence and early galectin‐1 upregulation in PIK3CA‐induced hepatocarcinogenesis: new therapeutic venues

Aberrant activation of the phosphoinositide 3‐kinase (PI3K)/AKT/mTOR and Ras/mitogen‐activated protein kinase (MAPK) pathways is a hallmark of hepatocarcinogenesis. In a subset of hepatocellular carcinomas (HCCs), PI3K/AKT/mTOR signaling dysregulation depends on phosphatidylinositol‐4,5‐bisphosphate 3‐kinase, catalytic subunit alpha (PIK3CA) mutations, while RAS/MAPK activation is partly attributed to promoter methylation of the tumor suppressor Ras association domain‐containing protein 1 (RASSF1A). To evaluate a possible cocarcinogenic effect of PIK3CA activation and RASSF1A knockout, plasmids expressing oncogenic forms of PIK3CA (E545K or H1047R mutants) were delivered to the liver of RASSF1A knockout and wild‐type mice by hydrodynamic tail vein injection combined with sleeping beauty‐mediated somatic integration. Transfection of either PIK3CA E545K or H1047R mutants sufficed to induce HCCs in mice irrespective of RASSF1A mutational background. The related tumors displayed a lipogenic phenotype with upregulation of fatty acid synthase and stearoyl‐CoA desaturase‐1 (SCD1). Galectin‐1, which was commonly upregulated in preneoplastic lesions and tumors, emerged as a regulator of SCD1. Co‐inhibitory treatment with PIK3CA inhibitors and the galectin‐1 inhibitor OTX008 resulted in synergistic cytotoxicity in human HCC cell lines, suggesting novel therapeutic venues.     

UBE2T promotes β‐catenin nuclear translocation in hepatocellular carcinoma through MAPK/ERK‐dependent activation

Ubiquitin‐conjugating enzyme E2T (UBE2T) has been implicated in many types of cancer including hepatocellular carcinoma (HCC). Epithelial–mesenchymal transition (EMT) process plays a fundamental role during tumor metastasis and progression. However, the molecular mechanisms underlying EMT in HCC in accordance with UBE2T still remain unknown. In this study, we showed that UBE2T overexpression augmented the oncogenic properties and specifically EMT in HCC cell lines, while its silencing attenuated them. UBE2T affected the activation of EMT‐associated signaling pathways: MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin. In addition, we revealed that the epithelial protein complex of E‐cadherin/β‐catenin, a vital regulator of signal transduction in tumor initiation and progression, was totally disrupted at the cell membrane. In particular, we observed that UBE2T overexpression led to E‐cadherin loss accompanied by a simultaneous elevation of both cytoplasmic and nuclear β‐catenin, while its silencing resulted in a strong E‐cadherin turnover at the cell membrane. Interestingly, chemical inhibition of the MAPK/ERK, AKT/mTOR, and Wnt/β‐catenin signaling pathways demonstrated that the nuclear translocation of β‐catenin and subsequent EMT was enhanced mainly by MAPK/ERK. Collectively, our findings demonstrate the UBE2T/MAPK‐ERK/β‐catenin axis as a critical regulator of cell state transition and EMT in HCC.     

Modifying chemotherapy response by targeted inhibition of eukaryotic initiation factor 4A

Translation is regulated predominantly at the initiation phase by several signal transduction pathways that are often usurped in human cancers, including the PI3K/Akt/mTOR axis. mTOR exerts unique administration over translation by regulating assembly of eukaryotic initiation factor (eIF) 4F, a heterotrimeric complex responsible for recruiting 40S ribosomes (and associated factors) to mRNA 5′ cap structures. Hence, there is much interest in targeted therapies that block eIF4F activity to assess the consequences on tumor cell growth and chemotherapy response. We report here that hippuristanol (Hipp), a translation initiation inhibitor that selectively inhibits the eIF4F RNA helicase subunit, eIF4A, resensitizes Eμ-Myc lymphomas to DNA damaging agents, including those that overexpress eIF4E—a modifier of rapamycin responsiveness. As Mcl-1 levels are significantly affected by Hipp, combining its use with the Bcl-2 family inhibitor, ABT-737, leads to a potent synergistic response in triggering cell death in mouse and human lymphoma and leukemia cells. Suppression of eIF4AI using RNA interference also synergized with ABT-737 in murine lymphomas, highlighting eIF4AI as a therapeutic target for modulating tumor cell response to chemotherapy.     

β-elemene suppresses tumor metabolism and stem cell-like properties of non-small cell lung cancer cells by regulating PI3K/AKT/mTOR signaling

Multi-drug resistance remains a critical issue in cancer treatment that hinders the effective use of chemotherapeutic drugs. The active components of traditional Chinese medicine have been applied as adjuvants to accentuate the anticancer properties of conventional drugs such as cisplatin. However, their application requires further validation and optimization. This study explored the anticancer activity of β-elemene, a natural component of traditional Chinese medical formulations. The effect of β-elemene on the anticancer properties of cisplatin was evaluated in A549 and NCI-H1650 lung cancer cells. Cell apoptosis, stem-like properties, glucose metabolism, multi-drug resistance, and PI3K/AKT/mTOR activation were assessed via flow cytometry, tumorsphere formation, and western blotting. The target genes of β-elemene were predicted using bioinformatics tools and validated in both cell lines. A xenograft model of lung cancer was established in nude mice to evaluate the combined effects of β-elemene and cisplatin in vivo. We found that β-elemene acted synergistically with cisplatin against non-small cell lung cancer cells by promoting apoptosis and impairing glucose metabolism, multi-drug resistance, and stemness maintenance. These effects were mediated by the inhibition of PI3K/AKT/mTOR activation. Bioinformatics analysis revealed that RB1 and TP53 are common target genes associated with lung cancer and β-elemene. The anti-tumorigenic properties of β-elemene were confirmed in vivo, wherein β-elemene, along with cisplatin, significantly suppressed tumor growth in a mouse xenograft model of non-small cell lung cancer. As such, β-elemene acted as an inhibitor of PI3K/AKT/mTOR signaling and enhanced the anticancer effect of cisplatin by targeting tumor metabolism, chemoresistance, and stem-like behavior. Thus, β-elemene is an effective anticancer adjuvant agent with potential clinical applications.     

Overexpression of the Long Noncoding RNA FOXD2-AS1 Promotes Cisplatin Resistance in Esophageal Squamous Cell Carcinoma Through the miR-195/Akt/mTOR Axis

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) mediate the development of esophageal squamous cell carcinoma (ESCC) via various pathophysiological pathways. This study explored the impact of the lncRNA FOXD2-AS1 on cisplatin resistance in ESCC and its possible mechanisms. Upregulation of FOXD2-AS was detected in patients with ESCC and ESCC cells that are resistant to cisplatin. In an in vitro assay, knockdown of FOXD2-AS1 noticeably inhibited cell invasion and growth, triggered cell death, and repressed the stimulation of the Akt/mTOR axis in cisplatin-resistant ESCC cells (TE-1/DDP). Conversely, the overexpression of FOXD2-AS1 remarkably increased cell invasion and growth, repressed cell death, and triggered the stimulation of the Akt/mTOR axis in TE-1/DDP cells. These findings, along with bioinformatics and validation tests, showed that FOXD2-AS1 targeted miR-195 by acting as a competing endogenous RNA. FOXD2-AS1/miR-195/Akt/mTOR axis plays a crucial role in resistance to cisplatin in ESCC cells, offering an innovative strategy to treat ESCC.     

circ_0006089 promotes gastric cancer growth,metastasis, glycolysis,and angiogenesis by regulating miR‐361‐3p/TGFB1

Circular RNA (circRNA) participates in a variety of pathophysiological processes, including the development of gastric cancer (GC). However, the role of circ_0006089 in GC progression and its underlying molecular mechanism need to be further revealed. Quantitative real‐time PCR was utilized for detecting circ_0006089, microRNA (miR)‐361‐3p and transforming growth factor‐β1 (TGFB1) expression. The interaction between miR‐361‐3p and circ_0006089 or TGFB1 was confirmed using a dual‐luciferase reporter assay and an RNA immunoprecipitation (RIP) assay. Cell proliferation, metastasis, apoptosis, and angiogenesis were determined using colony formation assay, EdU assay, transwell assay, flow cytometry, and tube formation assay. Cell glycolysis was evaluated by detecting glucose consumption, lactate production, and ATP levels. In addition, western blot (WB) analysis was used to measure protein expression. Xenograft tumor models were used to assess the effect of circ_0006089 knockdown on GC tumorigenesis. circ_0006089 had been found to be upregulated in GC tissues and cells, and it could act as an miR‐361‐3p sponge. circ_0006089 knockdown suppressed GC proliferation, metastasis, glycolysis, angiogenesis, and increased apoptosis, while this effect could be revoked by miR‐361‐3p inhibitor. TGFB1 was targeted by miR‐361‐3p, and its overexpression reversed the effects of miR‐361‐3p on GC cell function. Also, circ_0006089 promoted TGFB1 expression via sponging miR‐361‐3p. Animal experiments showed that silenced circ_0006089 inhibited GC tumorigenesis through the miR‐361‐3p/TGFB1 pathway. Our results revealed that the circ_0006089/miR‐361‐3p/TGFB1 axis contributed to GC progression, confirming that circ_0006089 might be a potential therapeutic target for GC.     

Circulating tumor DNA predicts efficacy of a dual AKT/p70S6K inhibitor (LY2780301) plus paclitaxel in metastatic breast cancer: plasma analysis of the TAKTIC phase IB/II study

The phosphatidylinositol‐3‐kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway is frequently activated in HER2‐negative breast cancer and may play a role in taxane resistance. The phase IB/II TAKTIC trial ({"type":"clinical-trial","attrs":{"text":"NCT01980277","term_id":"NCT01980277"}}NCT01980277) has shown that combining a dual AKT and p70 ribosomal protein S6 kinase (p70S6K) inhibitor (LY2780301) taken orally with weekly paclitaxel in HER2‐negative advanced breast cancer is feasible, with preliminary evidence of efficacy. We wanted to explore whether circulating tumor DNA (ctDNA) may be a surrogate marker of treatment efficacy in this setting. Serial plasma samples were collected and cell‐free DNA was sequenced using low‐coverage whole‐genome sequencing, and analysis was completed with droplet digital polymerase chain reaction (PCR) for some patients with driver mutations. Baseline tumor fraction (TF) and TF after 7 weeks on treatment were compared to progression‐free survival (PFS) and the overall response rate. We also explored circulating copy number alterations associated with treatment failure. Of the 51 patients enrolled in the TAKTIC trial, at least one plasma sample was available for 44 cases (96 timepoints). All patients with tumor TP53, PI3KCA, or AKT1 mutations harbored at least one of these alterations in plasma. TF at inclusion was correlated with PFS (6m‐PFS was 92% for ctDNAneg patients vs 68% for ctDNApos cases; hazard ratio [HR] = 3.45, 95% confidence interval [CI] [1.34–8.90], P = 0.007). ctDNA status at week 7 was not correlated with prognosis. Even though most circulating copy number alterations were conserved at disease progression, some genomic regions of interest were altered in post‐progression samples. In conclusion, ctDNA detection at baseline was associated with shorter PFS in patients included in the TAKTIC trial. Plasma‐based copy number analysis may help to identify alterations involved in resistance to treatment.     

Far‐infrared radiation alleviates cisplatin‐induced vascular damage and impaired circulation via activation of HIF‐1α

Severe vascular damage and complications are often observed in cancer patients during treatment with chemotherapeutic drugs such as cisplatin. Thus, development of potential options to ameliorate the vascular side effects is urgently needed. In this study, the effects and the underlying mechanisms of far‐infrared radiation (FIR) on cisplatin‐induced vascular injury and endothelial cytotoxicity/dysfunction in mice and human umbilical vein endothelial cells (HUVECs) were investigated. An important finding is that the severe vascular stenosis and poor blood flow seen in cisplatin‐treated mice were greatly mitigated by FIR irradiation (30 minutes/day) for 1‐3 days. Moreover, FIR markedly increased the levels of phosphorylation of PI3K and Akt, and VEGF secretion, as well as the expression and the activity of hypoxia‐inducible factor 1α (HIF‐1α) in cisplatin‐treated HUVECs in a promyelocytic leukemia zinc finger protein (PLZF)‐dependent manner. However, FIR‐stimulated endothelial angiogenesis and VEGF release were significantly diminished by transfection with HIF‐1α siRNA. We also confirmed that HIF‐1α, PI3K, and PLZF contribute to the inhibitory effect of FIR on cisplatin‐induced apoptosis in HUVECs. Notably, FIR did not affect the anticancer activity and the HIF‐1α/VEGF cascade in cisplatin‐treated cancer cells under normoxic or hypoxic condition, indicating that the actions of FIR may specifically target endothelial cells. It is the first study to demonstrate that FIR effectively attenuates cisplatin‐induced vascular damage and impaired angiogenesis through activation of HIF‐1α–dependent processes via regulation of PLZF and PI3K/Akt. Taken together, cotreatment with the noninvasive and easily performed FIR has a therapeutic potential to prevent the pathogenesis of vascular complications in cancer patients during cisplatin treatment.     

The Gene Expression Status of the PI3K/AKT/mTOR Pathway in Gastric Cancer Tissues and Cell Lines

The PI3K/AKT/mTOR pathway plays a crucial role in the regulation of multiple cellular functions including cell growth, proliferation, metabolism and angiogenesis. Emerging evidence has shown that deregulation of this pathway has a role promoting gastric cancer (GC). The aim was to assess the expression of genes involved in this pathway by qPCR in 23 tumor and 23 non-tumor gastric mucosa samples from advanced GC patients, and in AGS, MKN28 and MKN45 gastric cancer cell lines. Results showed a slight overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1, EIF4EBP1 and EIF4E genes, and a slightly decreased PTEN and TSC1 expression. In AGS, MKN28 and MKN45 cells a significant gene overexpression of PIK3CA, PIK3CB, AKT1, MTOR, RPS6KB1 and EIF4E, and a significant repression of PTEN gene expression were observed. Immunoblotting showed that PI3K-β, AKT, p-AKT, PTEN, mTOR, p-mTOR, P70S6K1, p-P70S6K1, 4E-BP1, p-4E-BP1, eIF4E and p-eIF4E proteins were present in cell lines at different levels, confirming activation of this pathway in vitro. This is the first time this extensive panel of 9 genes within PI3K/AKT/mTOR pathway has been studied in GC to clarify the biological role of this pathway in GC and develop new strategies for this malignancy.     

Long non‐coding RNA SNHG1 stimulates ovarian cancer progression by modulating expression of miR‐454 and ZEB1

Ovarian cancer (OC) is highly prevalent and is associated with high mortality rates due to metastasis and relapse. In this study, we assessed the role of long non‐coding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in OC to gain further insight into mechanisms that contribute to its aggressiveness. We analyzed the correlation between SNHG1, miR‐454 and zinc finger E‐box‐binding homeobox 1 (ZEB1) using a dual‐luciferase reporter assay. Alterations in cell metastasis and invasiveness were observed using wound‐healing and Transwell invasion assays, respectively. Tumor xenografts allowed us to monitor liver metastasis of mice injected with A2780 cells. We found that SNHG1 is overexpressed in OC. Downregulation of SNHG1 promoted miR‐454 expression and reduced ZEB1 levels. In addition, knockdown of SNHG1, also reduced the aggressiveness of A2780 and SK‐OV3 cells. Furthermore, SNHG1 downregulation by siRNA hindered cell migration and invasion; however, this effect was reversed by co‐transfection of miR‐454 into A2780 and SK‐OV3 cells. Moreover, SNHG1 increased ZEB1 expression by downregulating miR‐454 and activated Akt signaling, thereby promoting epithelial‐mesenchymal transition and enhancing the invasiveness of OC cells. Tumor xenograft analyses confirmed that SNHG1 affects OC proliferation and metastasis in vivo. In summary, our data demonstrate that SNHG1 plays crucial roles in tumor progression and may be a useful maker for OC prognosis.     

NGS‐based liquid biopsy profiling identifies mechanisms of resistance to ALK inhibitors: a step toward personalized NSCLC treatment

Despite impressive and durable responses, nonsmall cell lung cancer (NSCLC) patients treated with anaplastic lymphoma kinase (ALK) inhibitors (ALK‐Is) ultimately progress due to development of resistance. Here, we have evaluated the clinical utility of circulating tumor DNA (ctDNA) profiling by next‐generation sequencing (NGS) upon disease progression. We collected 26 plasma and two cerebrospinal fluid samples from 24 advanced ALK‐positive NSCLC patients at disease progression to an ALK‐I. These samples were analyzed by NGS and digital PCR. A tool to retrieve variants at the ALK locus was developed (VALK tool). We identified at least one resistance mutation in the ALK locus in ten (38.5%) plasma samples; the G1269A and G1202R mutations were the most prevalent among patients progressing to first‐ and second‐generation ALK‐Is, respectively. Overall, 61 somatic mutations were detected in 14 genes: TP53, ALK, PIK3CA, SMAD4, MAP2K1 (MEK1), FGFR2, FGFR3, BRAF, EGFR, IDH2, MYC, MET, CCND3, and CCND1. Specifically, a deletion in exon 19 in EGFR, a non‐V600 BRAF mutation (G466V), and the F129L mutation in MAP2K1 were identified in four patients who showed no objective survival benefit from ALK‐Is. Potential ALK‐I‐resistance mutations were also found in PIK3CA and IDH2. Finally, a c‐MYC gain, along with a loss of CCND1 and FGFR3, was detected in a patient progressing on a first‐line treatment with crizotinib. We conclude that NGS analysis of liquid biopsies upon disease progression identified different putative ALK‐I‐resistance mutations in most cases and could be a valuable approach for therapy decision making.     

Pseudogene KRT17P3 drives cisplatin resistance of human NSCLC cells by modulating miR‐497‐5p/mTOR

Chemoresistance is a major obstacle in non–small cell lung cancer (NSCLC) treatment. The pseudogene keratin 17 pseudogene 3 (KRT17P3) has been previously shown to be upregulated in lung cancer tissues of patients with cisplatin resistance. In the present study, RT‐qPCR was performed to evaluate KRT17P3 levels in plasma samples collected from 30 cisplatin‐resistant and 32 cisplatin‐sensitive patients. We found that the plasma level of KRT17P3 is upregulated in cisplatin‐resistant patients, and the increased expression of plasma KRT17P3 is associated with poor chemotherapy response. Functional studies demonstrated that KRT17P3 overexpression in cultured NSCLC cells increases cell viability and decreases apoptosis upon cisplatin treatment in vitro and in vivo, while KRT17P3 knockdown has the opposite effect. Mechanistically, bioinformatics analysis, RNA immunoprecipitation, and dual luciferase reporter assay indicated that KRT17P3 acts as a molecular sponge for miR‐497‐5p and relieves the binding of miR‐497‐5p to its target gene mTOR. Rescue experiments validated the functional interaction between KRT17P3, miR‐497‐5p, and mTOR. Taken together, our findings indicate that KRT17P3/miR‐497‐5p/mTOR regulates the chemosensitivity of NSCLC, suggesting a potential therapeutic target for cisplatin‐resistant NSCLC patients. KRT17P3 may be a potential peripheral blood marker of NSCLC patients resistant to cisplatin.     

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.     

Targeted inhibition of ERα signaling and PIP5K1α/Akt pathways in castration‐resistant prostate cancer

Selective ERα modulator, tamoxifen, is well tolerated in a heavily pretreated castration‐resistant prostate cancer (PCa) patient cohort. However, its targeted gene network and whether expression of intratumor ERα due to androgen deprivation therapy (ADT) may play a role in PCa progression is unknown. In this study, we examined the inhibitory effect of tamoxifen on castration‐resistant PCa in vitro and in vivo. We found that tamoxifen is a potent compound that induced a high degree of apoptosis and significantly suppressed growth of xenograft tumors in mice, at a degree comparable to ISA‐2011B, an inhibitor of PIP5K1α that acts upstream of PI3K/AKT survival signaling pathway. Moreover, depletion of tumor‐associated macrophages using clodronate in combination with tamoxifen increased inhibitory effect of tamoxifen on aggressive prostate tumors. We showed that both tamoxifen and ISA‐2011B exert their on‐target effects on prostate cancer cells by targeting cyclin D1 and PIP5K1α/AKT network and the interlinked estrogen signaling. Combination treatment using tamoxifen together with ISA‐2011B resulted in tumor regression and had superior inhibitory effect compared with that of tamoxifen or ISA‐2011B alone. We have identified sets of genes that are specifically targeted by tamoxifen, ISA‐2011B or combination of both agents by RNA‐seq. We discovered that alterations in unique gene signatures, in particular estrogen‐related marker genes are associated with poor patient disease‐free survival. We further showed that ERα interacted with PIP5K1α through formation of protein complexes in the nucleus, suggesting a functional link. Our finding is the first to suggest a new therapeutic potential to inhibit or utilize the mechanisms related to ERα, PIP5K1α/AKT network, and MMP9/VEGF signaling axis, providing a strategy to treat castration‐resistant ER‐positive subtype of prostate cancer tumors with metastatic potential.

Abbreviations

CRPC

castration‐resistant prostate cancer

DHT

dihydrotestosterone

E2

estradiol

ERα

estrogen receptor alpha

GO

gene ontology

NG‐CHM

Next‐Generation Clustered Heatmaps

PCa

prostate cancer

TMAs

tissue microarrays