Image_1_Immunochemotherapy achieved a complete response for metastatic adenocarcinoma of unknown primary based on gene expression profiling: a case report and review of the literature.tif

BackgroundCancer of unknown primary (CUP) is a malignant and aggressive tumor whose primary origin is still unknown despite thorough evaluation. CUP can be life-threatening with a median overall survival of less than 1 year based on empirical chemotherapy. Gene detection technology advances the driver gene detection of malignant tumors and the appropriate precise therapy. Immunotherapy has ushered in a new era in cancer therapy, changing the way advanced tumors, including CUP, are treated. Combined with comprehensive clinical and pathological investigations, molecular analysis of the original tissue and detection of potential driver mutations may provide therapeutic recommendations for CUP.Case presentationA 52-year-old female was admitted to hospital for dull abdominal pain, with peripancreatic lesions below the caudate lobe of the liver and posterior peritoneal lymph nodes enlargement. Conventional biopsy under endoscopic ultrasonography and laparoscopic biopsy both revealed poorly differentiated adenocarcinoma based on immunohistochemical series. To help identify tumor origin and molecular characteristics, 90-gene expression assay, tumor gene expression profiling with Next-generation sequencing (NGS) method and Immunohistochemical expression of PD-L1 were employed. Although no gastroesophageal lesions discovered by gastroenteroscopy, the 90-gene expression assay yielded a similarity score and prompted the most likely primary site was gastric/esophagus cancer. NGS revealed high TMB (19.3mutations/Mb) but no druggable driver genes identified. The Dako PD-L1 22C3 assay IHC assay for PD-L1 expression revealed a tumor proportion score (TPS) of 35%. Given the presence of negative predictive biomarkers for immunotherapy, including adenomatous polyposis coli (APC) c.646C>T mutation at exon 7 and Janus kinase 1(JAK1), the patient received immunochemotherapy instead of immunotherapy alone. She was successfully treated with nivolumab plus carboplatin and albumin-bound nanoparticle paclitaxel for six cycles and nivolumab maintenance, which achieved a complete response (CR) maintained for 2 years without severe adverse events.ConclusionsThis case highlights the value of multidisciplinary diagnosis and individual precision treatment in CUP. Further investigation is needed as an individualized treatment approach combining immunotherapy and chemotherapy based on tumor molecular characteristics and immunotherapy predictors is expected to improve the outcome of CUP therapy.</p

Additional file 1 of Genetic effect of metformin use on risk of cancers: evidence from Mendelian randomization analysis

Additional file 1. Table S1: SNPs associated with metformin use, which performed as instrumental variants (IVs) in two-sample MR analysis. Table S2: The genetic effect obtained from MVMR analysis. TT: total testosterone levels. Fig. S1: Scatter plots and funnel plots of metformin use on HER-positive breast cancer. A Scatter plots of the genetic association between metformin use and HER-positive breast cancer. The genetic predicted metformin use is associated with a lower risk of HER-positive breast cancer. The slope of each line shows the estimated causal effect of metformin use on HER-positive breast cancer for each approach. B Funnel plots showing the statistical association between metformin use and the risk of HER-positive breast cancer. Fig. S2: Leave-one-out analysis and Forest plots results. A Leave-one-out analysis of sensitivity test. After one by one eliminating the IVs, calculate the MR outcomes for the remaining IVs. B Forest plot of the causal effects of metformin use associated SNPs on HER-positive breast cancer. B Showed the Mendelian randomization estimated effects sizes for metformin use on HER-positive breast cancer. Fig. S3: Leave-one-out analysis result of metformin use on total testosterone levels. Fig. S4: Leave-one-out analysis result of total testosterone levels on HER-positive breast cancer. Fig. S5: Scatter plot of metformin use on total testosterone levels. Fig. S6: Scatter plot of total testosterone levels on HER-positive breast cancer. Fig. S7: Forest plot of metformin use on total testosterone levels. Fig. S8: Forest plot of total testosterone levels on HER-positive breast cancer. Fig. S9: Funnel plot of metformin use on total testosterone levels. Fig. S10: Funnel plot of total testosterone levels on HER-positive breast cancer

Genotype distributions and allele frequencies of five examined polymorphisms between lung cancer patients and controls, as well as the risk prediction under additive, dominant and recessive genetic models.

<p><i>Abbreviations</i>: OR, odds ratio; 95% CI, 95% confidence interval.</p>*<p>P values were adjusted for age, gender, smoking and drinking.</p><p>P_χ2 was calculated by χ² test for differences in genotypes and alleles between patients and controls.</p

The baseline characteristics of study population.

<p>Abbreviations: COPD, chronic obstructive pulmonary disease. Data are expressed as mean (standard deviation or SD) or percentage as indicated.</p>*<p>data not available.</p>**<p>P values were calculated by using unpaired t-test for age, and by χ2 test for other categorical characteristics.</p

TRIM59 regulates breast cancer cell actomyosin contractility and metastasis.

(A) Representative images of cell morphology changes upon TRIM59 deletion (T59 KO) in MCF7 cells (upper, left) or overexpression (T59 OE) in MDA-MB-231 cells (bottom, left). Scale bars: 20 μm. Quantification of the cell area of MCF7 (upper, right) and MDA-MB-231 cells (bottom, right). n = 6. (B) Representative confocal images of MCF7 cells depleted of TRIM59 and stained for F-actin, pSer19-MLC, and E-cadherin. Scale bars: 20 μm. (C) IB analysis of E-cadherin, pSer19-MLC expression in WT and TRIM59-KO MCF7 cells. (D) Representative confocal images of TRIM59 overexpressing (T59 OE) MDA-MB-231 cells with IRES-GFP stained for pSer19-MLC (red). Scale bars: 10 μm. (E) Representative confocal images of p-ERM expression (green) in WT and TRIM59-deleted MCF7 cells. Scale bars: 10 μm. (F) IB analysis of p-ERM and total ERM levels in WT, TRIM59 KD, or TRIM59 KO MCF7 cells (lanes 1–3) and WT or TRIM59 OE MDA-MB-231 cells (lanes 4–6). (G) Representative IHC staining images for p-ERM in tissue sections from xenograft tumors of TRIM59 KO MCF7 cells or TRIM59 OE MDA-MB-231 cells compared with control cells. Scale bars: 200 μm. (H) Spearman correlation between TRIM59 expression and ezrin phosphorylation (p-S148, p-S366) levels. (I) Representative images showing HE and CK8 staining of lung micro-metastases of xenograft tumors in the indicated groups (WT versus TRIM59 OE or TRIM59 KO in MCF7 or MDA-MB-231 cells). Scale bars: 100 μm (HE); 20 μm (CK8). Red arrow: pulmonary metastatic foci. Statistical analysis of the number of lung metastases was shown in the right panel. n = 6. (J and K) Representative luminescent images of the primary and metastases acquired with an IVIS imaging system. Luminescence imaging in NSG mice was followed after inoculation with breast cancer cells into the mammary fat-pad at the indicated time points (n = 3). Note, 10-week-old NSG mice inoculated with TRIM59 OE MDA-MB-231-luc died without bioluminescent imaging. (L) Gene expression analysis of TRIM59 in primary and lymph node metastases (GSE30480 dataset). (M) Representative TRIM59 IHC staining images in paired primary breast cancer and metastatic lymph nodes from breast cancer patients (n = 19). Statistical analysis of paired patient samples (primary versus metastatic tissues in the same patient) were shown on the right. Data in A, I, L, and M are presented as means ± SD. *P P S1 Data. CK8, cytokeratin 8; ERM, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558); HE, hematoxylin–eosin; IB, immunoblot; IHC, immunohistochemistry; IRES, internal ribosome entry site; GFP, green fluorescent protein; IVIS, in vivo imaging system; KD, knockdown; KO, knockout; Min, minimum; Max, maximum; NSG, NOD scid gamma; OE, overexpressed; p-ERM, phosphorylation of ERM; pSer19-MLC, phosphorylation of MLC at serine 19; TRIM59, tripartite motif 59; WT, wild-type.</p

Increased expression of TRIM59 correlates with breast cancer progression and poor survival in patients.

(A) The fold change of TRIM family genes across 12 cancer types (y-axis) compared with their paired controls (obtained from adjacent non-tumorigenic tissues) based on TCGA dataset analysis. The color intensity indicates the range of fold change, and black boxes indicate a significant difference in gene expression between tumor and adjacent non-tumorigenic tissues (fold change > 1.5 and P value TRIM family gene. Top hit TRIM59 was highlighted by red arrowhead. (B) Quantitative analysis of TRIM59 IHC staining in 87 paired tumor/non-tumor samples. (C) Representative IHC staining images of adjacent normal tissues and breast cancerous tissues probed with the anti-TRIM59 antibody. A total of 154 patient samples were stained and analyzed. (D) Analysis of TRIM59 staining intensity in association with clinical stages of breast tumor samples (Grade I, n = 18; Grade I–II, n = 26; Grade II and higher, n = 110). An IHC score less than or equal to 0.75 was defined as “Low IHC ratio” or Down-regulation/No change and a value greater than 0.75 was defined as “High IHC ratio” or Up-regulation. (E) Kaplan–Meier survival curves for breast cancer patients based on the scores of TRIM59 IHC staining. High intensity of TRIM59 immunostaining strongly associates with poor patient survival, P = 0.0244. Data in B and D are presented as means ± SD; *P P P S1 Data. BLCA, urothelial bladder cancer; BRCA, breast invasive carcinoma; COAD, colon adenocarcinoma; ESCA, esophageal carcinoma; HNSC, heck and neck squamous cell carcinoma; IHC, immunohistochemistry; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; STAD, stomach adenocarcinoma; TCGA, the Cancer Genome Atlas; THCA, thyroid carcinoma; TRIM, tripartite motif.</p

AIB1 regulates ERα-mediated SNAI1 expression.

<p>(A) Schematic illustration of ERα-binding elements in SNAI1 promoter. Fragments A to C were chosen for PCR amplification in ChIP assays. Three truncated versions of the SNAI1 promoter were made, and the length of each is shown in the illustration. (B) ChIP assays showing AIB1- and ERα-SNAI1 promoter interaction in T47D cells. Cross-linked chromatin was extracted from T47D cells and subjected to immunoprecipitation with anti-AIB1, anti-ERα or control IgG, and the resulting precipitated DNA was used as template for PCR-ampαlification of SNAI1 promoter using specific primer covering region A, B or C of the promoter region. (C) Reporter gene assays of different truncated versions of SNAI1 promoter in the presence of AIB1 or ERαoverexpression. Each of the truncated SNAI1 promoters was fused to luciferase gene in pGL3 and the resulting construct was introduced into T47D cells along with AIB1 or ERα construct or both. The levels of luciferase activity in these cells were determined 48 h after transfection. Luciferase activity was normalized to β-galactosidase activity, used to evaluate transfection efficiency. Each experiment was performed in triplicates and repeated at least of three times. Data are the means ± SDs. Statistically significant differences (<i>P</i><0.05) in paired Student’s t-test are marked with an asterisk.</p

TRIM59 depletion attenuates breast cancer cell survival and metastasis.

TRIM59 is essential for breast cancer cell mesenchymal movement and cell survival by maintaining low cell adhesion and high Wnt signaling of breast cancer cells. TRIM59 stabilizes PDCD10 by inhibiting K63 ubiquitination induced by RNFT1 at the lysine 179 site, and subsequently suppresses p62-selective autophagy degradation. TRIM59 deficiency facilitates PDCD10 degradation to overcome its inhibitory effect on Rho/ROCK signaling, thereby causing hyperactivated MLC and ERM and mesenchymal to amoeboid transition (MAT). TRIM59 deficiency also promotes excessive E-cadherin expression and reduces β-catenin expression, leading to high cell adhesion, low Wnt signaling, and high cell death to ultimately curtail tumor formation and metastasis. ERM, Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558); KO, knockout; K63-Ub, lysine 63-linked polyubiquitin; LC3, microtubule-associated protein 1A/1B-light chain 3; MAT, mesenchymal to amoeboid transition; MLC, myosin light chain; PDCD10, programmed cell death protein 10; p-ERM, phosphorylation of ERM; p-MLC, phosphorylation of MLC; p62, phosphotyrosine-independent ligand for the Lck SH2 domain of 62 kDa; Rho/ROCK, Ras homolog/Rho-associated coiled-coil kinase; RNFT1, RING finger and transmembrane domain-containing protein 1; TRIM59, tripartite motif 59; Ub, ubiquitin; WT, wild-type.</p

TRIM59 interacts with PDCD10 and blocks the autophagic degradation of PDCD10.

(A) Yeast two-hybrid screening to confirm the interaction between TRIM59-BD and PDCD10-AD. Transformed and mated cells were grown on–Leu–Trp medium (bottom). Interaction of TRIM59 with PDCD10 was verified on high-stringency plates (–Leu–Trp–His–Ade) and with a β-galactosidase filter assay and Aureobasidin A (AbA) (top). Interaction of p53 and SV40 large T-antigen (LTA) was used as positive control. (B) Co-IP and IB analysis of extracts of MCF7 cells with the indicated antibodies. IP with IgG was added as a negative control. (C) Co-IP and IB analysis of extracts of HEK293T cells transfected with FLAG-PDCD10 along with HA-tagged TRIM59 variants (full-length WT or deletions [ΔR, ΔTM, B, and CC] as shown on the top cartoon). (D) IB and qPCR analysis of PDCD10 protein expression (left) and mRNA levels (right) in shControl and shTRIM59 MCF7 cells (D, lane 1 versus 2) or in control and TRIM59 OE MDA-MB-231 cells (D, lane 3 versus 4). Data are presented as means ± SD. NS versus controls. The underlying data can be found in S1 Data. (E) Representative immunofluorescent staining of PDCD10 (green) in sections from xenograft of shControl or shTRIM59 MCF7 cells. DAPI, DNA-intercalating dye, indicates the nucleus (blue). Scale bars: 100 μm. n = 4. (F) IB analysis of PDCD10 expression in MCF7 WT or TRIM59 KO cell fractions. β-actin and LaminB1 were used as markers for the cytosolic and nuclear fractions, respectively. (G) IB analysis of PDCD10 expression in WT or TRIM59 KO MCF7 cells treated with DMSO or 10 μM MG132 (proteasome inhibitor) or 200 nM BafA1 (autophagy inhibitor). See S4C Fig for similar experiments using TRIM59 KD cells. ΔR, TRIM59 without RING domain; ΔTM, TRIM59 without the predicted transmembrane domain; AbA, Aureobasidin A; AD, transcription activation domain;–Ade, adenine dropout; B, B-box-type zinc finger domain; BafA1, bafilomycin A1; BD, DNA-binding domain; C, cytosolic fraction; CC, coiled-coil domain; co-IP, co-immunoprecipitation; HA, hemagglutinin;–His, histidine dropout; IB, immunoblot; IgG, immunoglobulin G; IP, immunoprecipitation; KD, knockdown; KO, knockout;–Leu, Leucine dropout; LTA, large T-antigen; N, nuclear fraction; NS, not significant; OE, overexpressing; PDCD10, programmed cell death protein 10; p53, tumor protein 53; qPCR, quantitative polymerase chain reaction; SV40, polyomavirus simian virus 40; TM, transmembrane domain; TRIM59, tripartite motif 59;–Trp, tryptophan dropout; WT, wild-type.</p

TRIM59 promotes breast cancer cell proliferation, growth, migration, and invasion in vitro.

(A) IB analysis of TRIM59 expression in the noncancerous mammary epithelial cell line (MCF10A) and breast cancer cell lines (MCF7 and MDA-MB-231). (B and C) IB analysis of TRIM59 expression in shRNA or CRISPR/Cas9 lentivirus-transduced control, TRIM59 KD (shTRIM59-2, KD efficiency confirmed by qPCR; B, middle) and TRIM59 KO MCF7 (B, left), or TRIM59-overexpressing (OE) MDA-MB-231 stable cells (C). sgRNA targeting TRIM59 was confirmed by Sanger sequencing (B, right). A dinucleotide GT insertion (underlined) caused an early abortion of TRIM59 translation, as further validated by immunoblotting (B, lane 4). (D and E) Graphic representation of 96-hour MTS proliferation assays of WT (non-transduced), control (transduced with non-targeting shRNA/sgRNA), shTRIM59 and TRIM59 KO MCF7 cells (D), and WT or TRIM59 OE MDA-MB-231 cells (E). (F and G) Colony formation assay in 3D Matrigel culture was performed to assess the rate of colony formation of control, shTRIM59, and TRIM59 KO MCF7 cells at day 8 (F) or WT and TRIM59 OE MDA-MB-231 cells at day 4 (G). Bar graphs show the number of colony-forming units (n = 8). Scale bars: 100 μm. (H and I) Scratched wound healing assays were performed to assess the rates of migration for control, shTRIM59, and TRIM59 KO MCF7 cells at 72 hours (H) or WT and TRIM59 OE MDA-MB-231 cells at 36 hours (I). Bar graphs show the percentage of migration rate (n = 8). Scale bars: 100 μm. (J and K) Transwell invasion assay was performed in Matrigel-coated 8-μm pore size transwell plates to assess the invasion ability of control, shTRIM59, and TRIM59 KO MCF7 cells (J) or WT and TRIM59 OE MDA-MB-231 cells (K). Bar graphs show the numbers of invaded cells (n = 8). Scale bars: 100 μm. Data in B, D-K are presented as means ± SD; *P P S1 Data. CRISPR/Cas9, clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease; IB, immunoblot; KD, knockdown; KO, knockout; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; OD, optical density; OE, overexpressing; qPCR, quantitative polymerase chain reaction; sgRNA, single guide RNA; shRNA, short hairpin RNA; TRIM59, tripartite motif 59; WT, wild-type.</p