ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

[Expression of fatty acid synthase and its association with HER2 in invasive ductal carcinoma of breast].

OBJECTIVE: To investigate the expression of fatty acid synthase (FAS) in adenosis, atypical ductal epithelial hyperplasia, ductal carcinoma in situ (DCIS) and invasive ductal carcinoma (IDC) of breast, and the correlation of FAS expression with HER2 gene amplification in IDC. METHODS: Immunohistochemical EnVision method staining for FAS was performed in 100 cases of breast lesions and 10 normal breast tissues. HER2 gene amplification was detected with FISH in 60 cases of IDC. RESULTS: The cohort included 10 cases of adenosis, 10 atypical ductal epithelial hyperplasia, 20 DCIS (8 high-grade, 9 intermediated-grade and 3 low-grade), and 60 cases of IDC (5 grade 1, 40 grade 2 and 15 grade 3). FAS expression was negative in all 10 normal breast tissues; in the 10 cases of adenosis, strongly positive FAS expression was detected in one case, positive in 2, weakly positive in 4, and negative in 3; in the 10 cases of atypical ductal epithelial hyperplasia, FAS immunohistochemistry showed that 1 was strongly positive, 4 positive, 4 weakly positive, and 1 negative; in the 20 cases of DCIS, FAS immunostaining showed that 12 were strongly positive, 5 positive, 1 weakly positive, and 2 negative; FAS expression showed a clear increasing trend from normal breast tissue, atypical ductal epithelial hyperplasia to DCIS (χ(2) = 42.02, P < 0.01). Likewise, the increasing trend was also demonstrated from adenosis to DCIS (χ(2) = 34.69, P < 0.01). There was also a positive correlation between FAS expression and extent of lesion among normal breast tissue, adenosis, atypical ductal epithelial hyperplasia and DCIS (χ(2) = 86.02, P < 0.01; r = 0.568, P < 0.01). FAS expression was not correlated with the grade of DCIS (χ(2) = 9.12, P = 0.16). In the five cases of grade 1 IDC, FAS immunostaining showed that 4 cases were strongly positive and 1 positive; in the 40 cases of grade 2 IDC, FAS immunostaining showed that 27 strongly positive, 12 positive, and 1 negative; in the 15 cases of grade 3 IDC, FAS immunostaining showed that 6 were strongly positive, 5 positive, 3 weakly positive, and 1 negative; FAS expression was stronger and more extensive in DCIS, IDC grades 1 and 2 than that in other groups. However, FAS expression was weaker in the IDC grade 3 (χ(2) = 11.26, P = 0.01). The positive expression rate of FAS in IDC was generally higher than that in benign breast lesions (χ(2) = 47.19, P < 0.01). In the 60 cases of IDC, FISH showed HER2 gene amplification in 22 cases, but not in the remaining 38 cases. FAS expression in IDC was highly correlated with HER2 gene amplification (r = 0.44, P < 0.01). The expression of FAS had significant correlation with status of ER and PR and tumor size (P < 0.05). There was no significant correlation with age, immunohistochemical HER2 expression, lymph node metastasis and clinical stage (P > 0.05). CONCLUSIONS: FAS may be closely related to the carcinogenesis of breast IDC. FAS expression is closely associated with HER2 gene amplification in IDC.

Deficiency of Tfh Cells and Germinal Center in Deceased COVID-19 Patients.

The COVID-19 pandemic caused by SARS-CoV2 is characterized by a remarkable variation in clinical severity ranging from a mild illness to a fatal multi-organ disease. Understanding the dysregulated human immune responses in the fatal subjects is critical for management of COVID-19 patients and the pandemic. In this study, we examined the immune cell compositions in the lung tissues and hilar lymph nodes using immunohistochemistry on 6 deceased COVID-19 patients and 4 focal organizing pneumonia (FOP) patients who underwent lung surgery and served as controls. We found a dominant presence of macrophages and a general deficiency of T cells and B cells in the lung tissues from deceased COVID-19 patients. In contrast to the FOP patients, Tfh cells and germinal center formation were largely absent in the draining hilar lymph nodes in the deceased COVID-19 patients. This was correlated with reduced IgM and IgG levels compared to convalescent COVID-19 patients. In summary, our data highlight a defect of germinal center structure in deceased COVID-19 patients leading to an impaired humoral immunity. Understanding the mechanisms of this deficiency will be one of the key points for the management of this epidemic.

[Effect of hypoxia on the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism].

OBJECTIVE: To explore the effect of hypoxia and hypoxia-inducible factor-1alpha (HIF-1alpha) on the expression of multidrug resistance gene-1 (mdr-1) and its coded p-glyeoprotein (P-gp) as well as the chemotherapeutic sensitivity of human ovarian cancer cells to paclitaxel and its mechanism. METHODS: The mRNA and protein levels of HIF-1alpha, mdr-1 and p-gp were studied by immunocytochemistry, semiquantitative reverse transcription-ploymerase chain reaction (RT-PCR) and Western blot analysis in human ovarian cancer cells (A2780) in 5% CO2 + 1% O2 hypoxic culture and 21% O2 normoxic culture, respectively. Methyl thiazolyl tetrazolium (MIT) was used to evaluate the chemotherapeutic sensitivity of A2780 cells to paclitaxel by inhibition rate. RNA interference technique was used and small hairpin RNAs (shRNAs) eukaryotic expression vector targeting HIF-1alpha was constructed as pSiHIF-1alpha, and transfected into A2780 cells. RT-PCR and Western blot were used to detect gene silencing effect on HIF-1alpha, the expressions of mdr-1 and p-gp. The inhibition rate was observed after HIF-1alpha gene silence. RESULTS: HIF-1alpha at both mRNA and protein levels was induced significantly under hypoxia. The HIF-1alpha expression at mRNA level was oxygen gradient-independent, while HIF-1alpha expression at protein level was oxygen gradient-dependent. The inhibition rate of paclitaxel to hypoxic A2780 cells in 5% CO2 + 1% O2 was significantly lower than that in normoxic A2780 cells (P <0.05). The shRNAs plasmid targeting HIF-1alpha was constructed successfully and HIF-1alpha gene was silenced in A2780 cells efficiently followed by mdr-1 and p-gp down-regulation. The inhibition rate was greatly increased in hypoxic A2780/siHIF-1alpha cells. CONCLUSION: Hypoxia can decrease the chemotherapeutic sensitivity of human ovarian cancer A2780 cells to paclitaxel through HIF-1alpha regulating the expression of mdr-1 and p-gp.

Cigarette smoke extract inhibits the proliferation of alveolar epithelial cells and induces apoptosis.

Cigarette smoke extract (CSE) contains abundant oxidants and free radicals. Oxidative stress caused by cigarette smoking results in the destruction of the alveolar cell walls and emphysema. However, there exists discrepancy about how CSE works in the process. In the present study, we observed the effect of CSE on the cell growth of type II alveolar epithelial cell-derived A549 cell line, and provided molecular understanding of this effect. The MTT assay results showed that CSE decreased the cell viability of A549 cells in a dose- and time-dependent manner, and cell cycle was arrested in G(1)/S phase. Furthermore, CSE-induced apoptosis of A549 cells was verified by Hoechst 33258 staining, electron microscopy in morphology, and the appearance of DNA fragmentation and annexin V-FITC/propidium iodide (PI) staining assay at molecular level. It was found that CSE treatment resulted in the upregulation of Fas/APO-1 receptor and activation of caspase-3. CSE also initiated accumulation of intracellular reactive oxygen species, which was detected by laser confocal microscopy. Taken together, CSE could inhibit the cell growth and induce apoptosis of A549 cells through Fas receptor pathway. Oxidative stress caused by CSE may be the radical factor leading to apoptosis as well as cell growth inhibition in alveolar epithelial cells.

[The role of myocardin in hypoxia-induced transdifferentiation of pulmonary artery endothelial cells into smooth muscle-like cells].

OBJECTIVE: To investigate the transdifferentiation of pulmonary artery endothelial cells (PAECs) into smooth muscle-like cells under hypoxia and the role of myocardin therein. METHODS: Recombinant plasmid psimyocardin (pSi), capable of silencing the expression of myocardin gene, was constructed by RNAi to be used to transfect the PAECs. Endothelial cells of adult pig pulmonary small arteries were purified by immunomagnetic purification technique, and divided into 4 groups: normoxia group (to be cultured in normoxic cell culture box containing 21% O(2), 5% CO2, and 74% N(2)), normoxia + pSi group, hypoxia group (to be cultured in cell box containing 1% O(2), 5% CO2, and 74% N(2)), and hypoxia + pSi group to be cultured for 1, 4, and 7 days respectively. Indirect fluorescence technique and morphological examination were used to identify the smooth muscle (SM) -like cells and the alpha-SM-actin positive cell ratio. RT-PCR was used to detect the mRNA expression of myocardin. RESULTS: Alpha-SM-actin positive cell could not be seen in the normoxia group and hypoxia 1 d group. The alpha-SM-actin positive cell rate of the hypoxia 7 days group was 2.07% +/- 0.06%, significantly higher than that of the hypoxia 4 d group (0.96% +/- 0.08%, P < 0.01). mRNA expression of myocardin gene could not be seen in the normoxia, normoxia + pSi, hypoxia 1 days, and hypoxia + pSi 1 days groups. The mRNA expression of myocardin gene of the hypoxia 7 days group was 0.23 +/- 0.03, significantly higher than that of the hypoxia 4 days group (0.14 +/- 0.01, P < 0.01). The mRNA expression levels of myocardin gene of the hypoxia + pSi 4 days and 7 days groups 0.03 +/- 0.02 and 0.05 +/- 0.01, both significantly lower than those of the hypoxia 4 days and 7 days groups respectively (0.14 +/- 0.01 and 0.23 +/- 0.03, both P < 0.01). The SM-like cell transdifferentiation rates of the hypoxia + pSi 4 days and 7 days groups were 0.19% +/- 0.07% and 0.21% +/- 0.04% respectively, both significantly lower than those of the corresponding hypoxia groups (0.96% +/- 0.08 and 2.07% +/- 0.06% respectively, both P < 0.01). CONCLUSION: Some PAECs have the potential to transdifferentiate into SM-like cells and may be one of the resources of muscularization of peripheral small vessels. Hypoxia remarkably promotes this transdifferentiation and myocardin may play an important role in this process.

[Impact of hypoxia on taxol-induced apoptosis in human ovarian cancer cell line A2780 and its mechanism].

BACKGROUND & OBJECTIVE: Hypoxia, a feature and important living microenvironment of solid tumors, might be related to drug-resistance of tumors. This study was to establish a hypoxic model of ovarian cancer cell line A2780, and to investigate impacts of hypoxia and hypoxia inducible factor-1alpha (HIF-1alpha) on Taxol-induced apoptosis in A2780 cells. METHODS: Cobalt chloride (CoCl(2)), a chemical hypoxia inducer, was added in A2780 cells to develop a hypoxic model. A2780 cells were divided into 4 groups: group A (control), group B (normoxia plus Taxol), group C (hypoxia plus Taxol), and group D (hypoxia, Decoy plus Taxol). Decoy method was used to block the function of HIF-1alpha. Protein and mRNA levels of HIF-1alpha were detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR). Cell apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL), and flow cytometry (FCM). RESULTS: CoCl(2) evidently increased protein level of HIF-1alpha, but had no effect on mRNA level of HIF-1alpha. Decoy didn't affect its expression. TUNEL showed that apoptotic index (AI) was significantly higher in group B,and group D than in group C after treatment of Taxol [(41.1+/-25.6)%,and (35.2+/-21.7)% vs. (24.1+/-15.2)%, P < 0.05]. The apoptosis rates detected by FCM displayed the same tendency as TUNEL results did. CONCLUSIONS: Hypoxia could weaken the effect of Taxol on inducing apoptosis of A2780 cells. HIF-1alpha might confer to resistance of cell apoptosis induced by Taxol.