N6-methyladenosine modified TGFB2 triggers lipid metabolism reprogramming to confer pancreatic ductal adenocarcinoma gemcitabine resistance.

Gemcitabine resistance is a major obstacle to the effectiveness of chemotherapy in pancreatic ductal adenocarcinoma (PDAC). Therefore, new strategies are needed to sensitize cancer cells to gemcitabine. Here, we constructed gemcitabine-resistant PDAC cells and analyzed them with RNA-sequence. Employing an integrated approach involving bioinformatic analyses from multiple databases, TGFB2 is identified as a crucial gene in gemcitabine-resistant PDAC and is significantly associated with poor gemcitabine therapeutic response. The patient-derived xenograft (PDX) model further substantiates the gradual upregulation of TGFB2 expression during gemcitabine-induced resistance. Silencing TGFB2 expression can enhance the chemosensitivity of gemcitabine against PDAC. Mechanistically, TGFB2, post-transcriptionally stabilized by METTL14-mediated m6A modification, can promote lipid accumulation and the enhanced triglyceride accumulation drives gemcitabine resistance by lipidomic profiling. TGFB2 upregulates the lipogenesis regulator sterol regulatory element binding factor 1 (SREBF1) and its downstream lipogenic enzymes via PI3K-AKT signaling. Moreover, SREBF1 is responsible for TGFB2-mediated lipogenesis to promote gemcitabine resistance in PDAC. Importantly, TGFB2 inhibitor imperatorin combined with gemcitabine shows synergistic effects in gemcitabine-resistant PDAC PDX model. This study sheds new light on an avenue to mitigate PDAC gemcitabine resistance by targeting TGFB2 and lipid metabolism and develops the potential of imperatorin as a promising chemosensitizer in clinical translation.

CSNK2A1 confers gemcitabine resistance to pancreatic ductal adenocarcinoma via inducing autophagy.

Gemcitabine, a pivotal chemotherapeutic agent for pancreatic ductal adenocarcinoma (PDAC), frequently encounters drug resistance, posing a significant clinical challenge with implications for PDAC patient prognosis. In this study, employing an integrated approach involving bioinformatic analyses from multiple databases, we unveil CSNK2A1 as a key regulatory factor. The patient-derived xenograft (PDX) model further substantiates the critical role of CSNK2A1 in gemcitabine resistance within the context of PDAC. Additionally, targeted silencing of CSNK2A1 expression significantly enhances sensitivity of PDAC cells to gemcitabine treatment. Mechanistically, CSNK2A1's transcriptional regulation is mediated by H3K27 acetylation in PDAC. Moreover, we identify CSNK2A1 as a pivotal activator of autophagy, and enhanced autophagy drives gemcitabine resistance. Silmitasertib, an established CSNK2A1 inhibitor, can effectively inhibit autophagy. Notably, the combinatorial treatment of Silmitasertib with gemcitabine demonstrates remarkable efficacy in treating PDAC. In summary, our study reveals CSNK2A1 as a potent predictive factor for gemcitabine resistance in PDAC. Moreover, targeted CSNK2A1 inhibition by Silmitasertib represents a promising therapeutic strategy to restore gemcitabine sensitivity in PDAC, offering hope for improved clinical outcomes.

MFAP5 facilitates the aggressiveness of intrahepatic Cholangiocarcinoma by activating the Notch1 signaling pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. The dismal outcome of ICC patients is due to lack of early diagnosis, the aggressive biological behavior of ICC and the lack of effective therapeutic options. Early diagnosis and prognosis of ICC by non-invasive methods would be helpful in providing valuable information and developing effective treatment strategies. METHODS: Expression of microfibrillar-associated protein 5 (MFAP5) in the serum of ICC patients was detected by ELISA. Human ICC specimens were immunostained by MFAP5 antibodies. The growth rate of human ICC cell lines treated with MFAP5 or MFAP5 shRNAs was examined by CCK8 and colony formation assays. Cell cycle analysis was performed with PI staining. The effect of MFAP5 inhibition was assessed by xenograft models in nude mice. RNA-seq and ATAC-seq analyses were used to dissect the molecular mechanism by which MFAP5 promoted ICC aggressiveness. RESULTS: We identified MFAP5 as a biomarker for the diagnosis and prognosis of ICC. Upregulated MFAP5 is a common feature in aggressive ICC patients' tissues. Importantly, MFAP5 level in the serum of ICC patients and healthy individuals showed significant differential expression profiles. Furthermore, we showed that MFAP5 promoted ICC cell growth and G1 to S-phase transition. Using RNA-seq expression and ATAC-seq chromatin accessibility profiling of ICC cells with suppressed MFAP5 secretion, we showed that MFAP5 regulated the expression of genes involved in the Notch1 signaling pathway. Furthermore, FLI-06, a Notch signaling inhibitor, completely abolished the MFAP5-dependent transcriptional programs. CONCLUSIONS: Raised MFAP5 serum level is useful for differentiating ICC patients from healthy individuals, and could be helpful in ICC diagnosis, prognosis and therapies.

[Blocking programmed death-ligand 1 attenuates maturation inhibition of dendritic cells by co-cultured breast cancer cells].

OBJECTIVE: To study if programmed death-ligand 1 (PL-L1) expression in breast cancer cell activates PD-L1/PD-1 pathway in dendritic cells to inhibit dendritic cell maturation. METHODS: Human monocytes were induced to differentiate into immature dendritic cells using GM-CSF and IL-4, and further to mature dendritic cells using TNF-α. PD-L1-expressing breast cancer cell line MDA-MB-231 was co-cultured in contact with the dendritic cells to observe the effects of the breast cancer cells on the maturation of the dendritic cells. A PD-L1 blocking antibody was applied to the co-culture, and the changes in the inhibitory effect of the MDA-MB-231 cells on dendritic cell maturation was observed. TNF-α-induced dendritic cells were treated with a recombinant human PD-L1 protein to study the effect of PD-L1/PD-1 pathway activation on the maturation of dendritic cells. The expression of PD-L1 in MDA-MB-231 cells and the dendritic cell maturation marker HLA-DR and CD83 were analyzed using flow cytometry. RESULTS: MDA-MB-231 cell line showed PD-L1 positivity on the cell membrane cells at a rate as high as (99.7∓0.15)%. In mature dendritic cells, the positivity rates for HLA-DR and CD83 were (88.8∓6.96)% and (18.36∓3.07)%, respectively, but in the co-culture system, the positivity rates of the dendritic cells were significantly decreased to (42.76∓10.52)% (P<0.01) and (9.93∓2.74)% (P<0.05), respectively, indicating that MDA-MB-231 cells inhibited the maturation of dendritic cells. Following treatment with a PD-L1 antibody isotype control, the percentages of HLA-DR- and CD83-positive cells in the co-culture were (45.17∓10.19)% and (10.15∓2.54)%, which were significantly increased to (63.46∓1.72)% and (16.46∓2.58)% after treatment with PD-L1 antibody, respectively (both P<0.05). Compared with the mature dendritic cell controls, the cells treated with the recombinant human PD-L1 protein exhibited significantly lowered percentages of HLA-DR-positive [from (84.23∓4.18)% to (2.56∓2.39)%, P<0.05] and CD83-positive cells [(87.26∓1.54)% to (60.67∓1.63)%, P<0.05]. CONCLUSION: The effect of PD-L1 antibody therapy on triple negative breast cancer can be partially mediated by blocking PD-L1 expression on breast cancer cell membrane, which attenuates the inhibition of dendritic cell maturation in the cancer microenvironment.

[Phosphorylation of ezrin Tyr477 is critical in invasion of breast cancer cells stimulated by precursor of nerve growth factor].

OBJECTIVE: To investigate the effect of precursor of nerve growth factor (proNGF) in promoting invasion of breast cancer cells and its relation with ezrin expression and phosphorylation of ezrin Thr567 and Tyr477. METHODS: Human breast cancer cell lines MDA-MB-231 and MCF-7 were stimulated by gradient concentrations of proNGF (0, 2.5, 5 and 10 ng/mL) for 16 h, and the invasion of the cells was assessed with Transwell assay. The expression of ezrin and the phosphorylation of ezrin Thr567 and ezrin Tyr477 in the treated cells were examined by Western blotting. MDA-MB-231 cells were transfected with pEnter-His-ezrinY477F (a dominant negative mutant) to study the role of phosphrylation of ezrin Tyr477 in the invasion of breast cancer cell stimulated by proNGF. RESULTS: proNGF significantly promoted MDA-MB-231 and MCF-7 cell invasion in a concentration-dependent manner (P<0.05), and concentration- and time-dependently increased the phosphorylation of ezrin Tyr477 (P<0.05) without affecting the expression of ezrin or the phosphorylation of ezrin Thr567. The specific inhibitor of src, SKI-606, significantly inhibited the phosphorylation of ezrin Tyr477 induced by proNGF. Transfection with pEnter-His- ezrinY477F inhibited proNGF-induced invasion and phosphorylation of ezrin Tyr477 in MDA-MB-231 cells (P<0.05). CONCLUSION: Phosphorylation of ezrin Tyr477 plays a critical role in the invasion of breast cancer cells stimulated by proNGF via proNGF/src/ezrin Tyr477 pathway.

[Nanog promotes the invasion of breast cancer cells by increasing PKCε expression].

OBJECTIVE: To study the relationship between Nanog-promoted metastasis of breast cancer and ezrin(T567) phosphorylation, and explore the possible mechanism by which Nanog regulates ezrin(T567) phosphorylation. METHODS: A siRNA construct targeting Nanog was transfected in breast cancer cells to knock down Nanog expression, and the changes in the cell invasion was detected using Transwell assay. The expression levels of Nanog and PKC and the phosphorylation level of ezrin(T567) were detected using Western blotting and immunofluorescent staining; the protein interaction between PKCε and ezrin was assayed by co-immunoprecipitation and Western blotting. RESULTS: Nanog knockdown significantly decreased the expression of PKCε protein, phosphorylation level of ezrin(T567) and the invasion ability of breast cancer cells. PKCε knockdown obviously decreased the phosphorylation level of ezrin(T567) in the cells, and PKCε and ezrin were co-immunoprecipitated. CONCLUDIONS: Nanogcan can upregulate the expression of PKCε to promote the phosphorylation of ezrin(T567), which can be a new mechanism by which Nanog promotes tumor metastasis.

[Tricostantin A inhibits self-renewal of breast cancer stem cells in vitro].

OBJECTIVE: To investigate the effect of tricostantin A (TSA) on self-renewal of breast cancer stem cells and explore the mechanisms. METHODS: Breast cancer cell lines MDA-MB-468, MDA-MB-231, MCF-7 and SKBR3 were cultured in suspension and treated with different concentrations of TSA for 7 days, using 0.1% DMSO as the control. Secondary mammosphere formation efficiency and percentage of CD44(+)/CD24(-) sub-population in the primary mammospheres were used to evaluate the effects of TSA on self-renewal of breast cancer stem cells. The breast cancer stem cell surface marker CD44(+)/CD24(-) and the percentage of apoptosis in the primary mammospheres were assayed using flow cytometry. The mRNA expressions of Nanog, Sox2 and Oct4 in the primary mammospheres were assayed with quantitative PCR. RESULTS: TSA at both 100 and 500 nmol/L, but not at 10 nmol/L, partially inhibited the self-renewal of breast cancer stem cells from the 4 cell lines. TSA at 500 nmol/L induced cell apoptosis in the primary mammospheres. TSA down-regulated the mRNA expression of Nanog and Sox2 in the primary mammospheres. CONCLUSION: TSA can partially inhibit the self-renewal of breast cancer stem cells through a mechanism involving the down-regulation of Nanog and Sox2 expression, indicating the value of combined treatments with low-dose TSA and other anticancer drugs to achieve maximum inhibition of breast cancer stem cell self-renewal. The core transcriptional factor of embryonic stem cells Nanog and Sox2 can be potential targets of anticancer therapy.