Phosphoproteomic analysis of metformin signaling in colorectal cancer cells elucidates mechanism of action and potential therapeutic opportunities.

BACKGROUND: The biguanide drug metformin is a safe and widely prescribed drug for type 2 diabetes. Interestingly, hundreds of clinical trials have been set to evaluate the potential role of metformin in the prevention and treatment of cancer including colorectal cancer (CRC). However, the "metformin signaling" remains controversial. AIMS AND METHODS: To interrogate cell signaling induced by metformin in CRC and explore the druggability of the metformin-rewired phosphorylation network, we performed integrative analysis of phosphoproteomics, bioinformatics, and cell proliferation assays on a panel of 12 molecularly heterogeneous CRC cell lines. Using the high-resolute data-independent analysis mass spectrometry (DIA-MS), we monitored a total of 10,142 proteins and 56,080 phosphosites (P-sites) in CRC cells upon a short- and a long-term metformin treatment. RESULTS AND CONCLUSIONS: We found that metformin tended to primarily remodel cell signaling in the long-term and only minimally regulated the total proteome expression levels. Strikingly, the phosphorylation signaling response to metformin was highly heterogeneous in the CRC panel, based on a network analysis inferring kinase/phosphatase activities and cell signaling reconstruction. A "MetScore" was determined to assign the metformin relevance of each P-site, revealing new and robust phosphorylation nodes and pathways in metformin signaling. Finally, we leveraged the metformin P-site signature to identify pharmacodynamic interactions and confirmed a number of candidate metformin-interacting drugs, including navitoclax, a BCL-2/BCL-xL inhibitor. Together, we provide a comprehensive phosphoproteomic resource to explore the metformin-induced cell signaling for potential cancer therapeutics. This resource can be accessed at https://yslproteomics.shinyapps.io/Metformin/.

Comprehensive analysis of PTPN gene family revealing PTPN7 as a novel biomarker for immuno-hot tumors in breast cancer.

Background: The non-receptor protein tyrosine phosphatase (PTPN) gene family has been considered to be involved in the oncogenesis and development of multiple cancers. However, its prognostic utility and immunological relevance in breast cancer (BrCa) have not been clarified. Methods: A transcriptional level interpretation of the expressions and prognostic values was analyzed using the data from The Cancer Genome Atlas (TCGA) cohort. In addition, GO and DAVID pinpoint the functional enrichment of PTPNs. Moreover, the immune correlations of PTPN7 in BrCa and pan-cancer were further investigated based on the TCGA cohort and were testified using the in-house and the Gene Expression Omnibus (GEO) cohorts. Results: For systematic analysis of the PTPN family, we found that the expression levels of PTPN1, PTPN6, PTPN7, PTPN18, PTPN20, and PTPN22 was promoted in tumor tissues while comparing with paraneoplastic tissues during our study. We further investigated their functions and protein-protein interactions (PPI), and these results strongly suggested that PTPN family was associated with protein dephosphorylation. Next, we performed an immunological relevance analysis and found that PTPN7 was correlated with immune infiltration, suggesting a stronger association of PTPN7 with immuno-hot tumors in BrCa. In addition, results from the in-house cohort confirmed the positive correlation between PTPN7 and PD-L1. The pan-cancer analysis revealed that PTPN7 was related to PD-L1 and CTLA-4 expression in almost all cancer types. Finally, the predictive value of PTPN7 for immunotherapy was significant in two independent GEO cohorts. Conclusion: In conclusion, this is the first extensive research on the correlation between PTPN family expression and immune characterization in BrCa. As results, PTPN7 expression is associated with immuno-hot tumors and could be a promising predictive biomarker for immunotherapy in not only BrCa but multiple cancers.

Systematic Characterization of Expression Patterns and Immunocorrelations of Formin-Like Genes in Breast Cancer.

Background: Members of the formin-like gene (FMNL) family are required for cytoskeleton-related processes, and their expressions are implicated to the progression of a multitude of malignancies. However, there are insufficient studies on transcription factors and promising prognosis benefit of FMNLs during the genesis of breast cancer (BrCa). Methods: The transcriptional levels of FMNL family members in primary BrCa tissues and their association with intrinsic subclasses were analyzed using the UALCAN database. Then, the prognostic values of FMNLs in BrCa patients were investigated via the Kaplan-Meier plotter. Moreover, the correlations between FMNL expression levels and immune infiltrations were analyzed using the TIMER database. In addition, the expression patterns of FMNLs in BrCa were investigated by single-cell RNA-sequencing (scRNA-seq) analysis and were validated by immunohistochemistry (IHC) staining. Results: The transcriptional level of FMNL1 was shown to be considerably increased in BrCa. It is surprising that the transcriptional quantities of FMNL2 and FMNL3 were substantially reduced. In addition, during the comparison of several BrCa subclasses, FMNL1 and FMNL2 mRNA levels of patients with HER2-positive and triple-negative BrCa subclasses increased, while FMNL3 mRNA levels reduced. With the processions of experimentation, high FMNL1 expression was hopefully linked to well clinical outcome, while high FMNL2 expression predicted poor prognosis. Moreover, FMNL1 was highly expressed in tumor-infiltrating immune cells (TIICs) in tumor tissues. Last but not least, FMNL1 was highly expressed in TIICs and served as a gene marker for TIICs. Conclusions: The fact and result which we analyzed demonstrate FMNL1 as a diagnostic marker for TIICs by comprehensively elucidating the expression patterns and changeable prognostic implications of FMNLs in BrCa clinical applications.

Overexpression of CDCP1 is Associated with Poor Prognosis and Enhanced Immune Checkpoints Expressions in Breast Cancer.

CUB-domain containing protein 1 (CDCP1) is a transmembrane protein acting as an effector of SRC family kinases, which play an oncogenic role in multiple human cancers. However, its clinical and immune correlations in breast cancer (BrCa) have not been explored. To define the expression, prognostic value, and potential molecular role of CDCP1 in BrCa, multiple public datasets, and an in-house cohort were used. Compared with paratumor tissue, CDCP1 was remarkably upregulated in the tumor tissues at both mRNA and protein levels. In the in-house cohort, CDCP1 protein expression was related to several clinicopathological parameters, including age, ER status, PR status, molecular type, and survival status. Kaplan-Meier analysis and Cox regression analysis exhibited that CDCP1 was an important prognostic biomarker in BrCa. In addition, enrichment analysis uncovered that CDCP1 was not only involved in multiple oncogenic pathways, but correlated with overexpression of immune checkpoints. Overall, we reported that increased expression of CDCP1 is a favorable prognostic factor in patients with BrCa. In addition, the correlations between CDCP1 and immune checkpoints provide a novel insight into the adjuvant treatment for immune checkpoint blockade via targeting CDCP1.

Data-independent acquisition-based proteome and phosphoproteome profiling across six melanoma cell lines reveals determinants of proteotypes.

Human cancer cell lines are widely used in pharmacological and systems biological studies. The rapid documentation of the steady-state gene expression landscape of the cells used in a particular experiment may help to improve the reproducibility of scientific research. Here we applied a data-independent acquisition mass spectrometry (DIA-MS) method, coupled with a peptide spectral-library-free data analysis workflow, to measure both the proteome and phosphoproteome of a melanoma cell line panel with different metastatic properties. For each cell line, the single-shot DIA-MS detected 8100 proteins and almost 40 000 phosphopeptides in the respective measurements of two hours. Benchmarking the DIA-MS data towards the RNA-seq data and tandem mass tag (TMT)-MS results from the same set of cell lines demonstrated comparable qualitative coverage and quantitative reproducibility. Our data confirmed the high but complex mRNA-protein and protein-phospsite correlations. The results successfully established DIA-MS as a strong and competitive proteotyping approach for cell lines. The data further showed that all subunits of the glycosylphosphatidylinositol (GPI)-anchor transamidase complex were overexpressed in metastatic melanoma cells and identified altered phosphoprotein modules such as the BAF complex and mRNA splicing between metastatic and primary cells. This study provides a high-quality resource for calibrating DIA-MS performance, benchmarking DIA bioinformatic algorithms, and exploring the metastatic proteotypes in melanoma cells.

Global and Site-Specific Effect of Phosphorylation on Protein Turnover.

To date, the effects of specific modification types and sites on protein lifetime have not been systematically illustrated. Here, we describe a proteomic method, DeltaSILAC, to quantitatively assess the impact of site-specific phosphorylation on the turnover of thousands of proteins in live cells. Based on the accurate and reproducible mass spectrometry-based method, a pulse labeling approach using stable isotope-labeled amino acids in cells (pSILAC), phosphoproteomics, and a unique peptide-level matching strategy, our DeltaSILAC profiling revealed a global, unexpected delaying effect of many phosphosites on protein turnover. We further found that phosphorylated sites accelerating protein turnover are functionally selected for cell fitness, enriched in Cyclin-dependent kinase substrates, and evolutionarily conserved, whereas the glutamic acids surrounding phosphosites significantly delay protein turnover. Our method represents a generalizable approach and provides a rich resource for prioritizing the effects of phosphorylation sites on protein lifetime in the context of cell signaling and disease biology.

Prognostic value of the systemic inflammation response index (SIRI) before and after surgery in operable breast cancer patients.

BACKGROUND: The new systemic inflammation response index (SIRI) constructed based on neutrophil, monocyte and lymphocyte counts in peripheral blood is considered to be related to the prognosis of a variety of tumours. OBJECTIVE: To evaluate the prognostic value of the SIRI in operable breast cancer patients and establish a nomogram to predict the survival of breast cancer patients. METHODS: A total of 949 patients with operable breast cancer were enrolled in the present study. RESULTS: The overall survival (OS) of breast cancer patients with SIRI ⩽ 0.65 was significantly higher than that of breast cancer patients with SIRI > 0.65 (P< 0.001). A nomogram generated based on SIRI, grade and TNM stage and SIRI predicted the 5- and 10-year survival rates of breast cancer patients more accurately than TNM stage alone. In addition, the change in SIRI relative to baseline at 4 weeks after surgery was closely related to the survival of breast cancer patients. Compared with those with no SIRI changes (absolute value of variation < 25%), breast cancer patients with an increase in SIRI > 75% or 25-75% had worse OS (P< 0.001). CONCLUSIONS: The SIRI before and after surgery is closely related to the prognosis of breast cancer patients.

Increased prediction of right nonrecurrent laryngeal nerve in thyroid surgery using preoperative computed tomography with intraoperative neuromonitoring identification.

BACKGROUND: A nonrecurrent laryngeal nerve (NRLN) is a rare but potentially serious anatomical variant. Although the incidence is reported to be 0.3% to 1.3%, it carries a much higher risk of palsy during thyroid surgery. The objective of this study is to investigate the usefulness of computed tomography (CT) for preoperative identification and intraoperative neuromonitoring identification (IONM) of NRLN in thyroid cancer patients. METHODS: The preoperative neck CT scans from 1,574 patients who needed thyroid surgery were examined. Absence of the brachiocephalic artery (BCA) and the presence of arteria lusoria were defined as positive with NRLN. Systematic intraoperative neuromonitoring (IONM) was also carried out for these 1,574 patients to localize and identify NRLN. A negative electromyography (EMG) response from lower vagal stimulation but a positive EMG response from the upper position indicated the occurrence of an NRLN. RESULTS: Nine NRLN (0.57%) were intraoperatively identified out of the 1,574 patients, and no patient with a NRLN showed preoperative clinical symptoms related to NRLN. Prior to the operation, surgeons identified only seven suspected NRLN cases based on identification of arteria lusoria. But a review of CT scans revealed that all cases could be identified by vascular anomalies. All patients were successfully detected at an early stage of operation using intraoperative neuromonitoring (IONM). Postoperative vocal cord function was normal in all patients. CONCLUSIONS: CT of the neck is a reliable method for predicting NRLN before thyroid cancer surgery. However, some image features can be easily missed. Neurophysiology helps the surgeon to identify the NRLNs more precisely. Combining the two evaluation methods may decrease the incidence of nerve palsy, especially in cases of NRLN. Considering that CT is expensive, requires an X-ray, and achieves less information than ultrasound (US) concerning thyroid nodules, we suggest that applying US and IONM is more reasonable.

Number of central lymph node metastasis for predicting lateral lymph node metastasis in papillary thyroid microcarcinoma.

BACKGROUND: The purpose of this study was to present our evaluation of the relationship between the number of positive central lymph nodes and lateral lymph node metastasis in patients with papillary thyroid microcarcinoma (PTMC). METHODS: Up to 141 patients with PTMC were divided into 3 groups according to different positive central lymph node classifications as follows: group A, no positive central lymph node; group B, 1 positive central lymph node; and group C, 2 or more positive central lymph nodes. RESULTS: Incidence of lateral lymph node metastasis was 30.5% (43 of 141). It was significantly high in group C compared with groups A and B, although there was no significant difference between groups A and B. Number of positive central lymph node ≥2, underlying Hashimoto thyroiditis, and extrathyroidal extension were the independent predictive factors for lateral lymph node metastasis on multivariate analysis. CONCLUSION: Lateral lymph node metastasis was mainly observed in patients with ≥2 positive central lymph nodes, which is an independent predictive factor for lateral lymph node metastasis. Therefore, ≥2 positive central lymph nodes may be valuable in predicting lateral lymph node metastasis.

Effectiveness of OK-432 (Sapylin) to reduce seroma formation after axillary lymphadenectomy for breast cancer.

BACKGROUND: The occurrence of seroma formation after axillary lymphadenectomy for breast cancer cannot be ignored. Various approaches have been used in an effort to reduce it, but these results are still controversial. We aimed to describe a new method of application of OK-432 (Sapylin, heat-treated Su strain of Streptococcus) to reduce seroma formation after axillary lymphadenectomy for breast cancer and to verify the safety and efficacy of it as a beneficial supplement for conventional surgery. METHODS: A prospective, randomized analysis of consecutive quadrantectomy or mastectomy plus axillary lymphadenectomy using or not using OK-432 was designed. From July 2010 to November 2011, a total of 111 patients were enrolled in this prospective, randomized study and completed the follow-up. OK-432 applied to the axillary fossa plus placement of closed suction drainage was used in 54 patients (the experimental group); placement of closed suction drainage was used in 57 patients (the control group). RESULTS: There were no statistical significance between the two groups in terms of age, body mass index, treatment received, tumor size, number of removed lymph nodes, and lymph node status. Postoperative drainage magnitude and duration were significantly reduced in the experimental group (P = 0.008 and 0.003, respectively). One week after hospital discharge, fewer patients developed a palpable seroma in the experimental group: 10 in the experimental group versus 28 in the control group (P = 0.001). Fewer seromas needed aspiration (mean 1 [range 0-3] in the experimental group vs. mean 4 [range 1-5] in the control group; P < 0.001). There were no significant differences in terms of the incidence of complications associated with axillary lymphadenectomy (P = 0.941). CONCLUSIONS: OK-432 is a feasible and safe option for axillary lymphadenectomy for breast cancer. The use of it does not always prevent seroma formation, but it can reduce drainage magnitude and duration, as well as decrease the incidence of seroma after the removal of drainage. It may be increasingly conducted in day surgery clinics.

Different distribution of breast ductal carcinoma in situ, ductal carcinoma in situ with microinvasion, and invasion breast cancer.

BACKGROUND: Breast ductal cancer in situ (DCIS) can recur or progress to invasive ductal cancer (IDC), and the interim stage include DCIS with microinvasion (DCIS-Mi). In this article, we attempt to study the study the differences of clinicopathological features, imaging data, and immunohistochemical-based subtypes among DCIS, DCIS-Mi, and IDC. METHODS: In this retrospective study, we attempt to compare the clinicopathological features, immunohistochemical results and imaging data of 866 patients (included 73 DCIS, 72 DCIS-Mi, and 721 IDC). RESULTS: Patients with DCIS and DCIS-Mi were younger than those with IDC (P = 0.007). DCIS and DCIS-Mi often happened in premenopausal women while IDC was opposite (P <0.001). The incidence of IDC with node-positive was significantly higher than it in DCIS and DCIS-Mi (P <0.001). We also observed that the Her2-positive was more often found in patients with pure DCIS compared to those with DCIS-Mi and DCIS-I (P <0.001). There was a significant difference between the four subgroups (Luminal-A, Luminal-B, ERBB2+, Basal-like) from DCIS, DCIS-Mi, and IDC (P <0.001). Basal-like patients were fewer than other subgroups in DCIS, DCIS-Mi, and IDC. The incidence of the first performance of ultrasound (catheter winded and nodular mass) and mammography (nodular mass) had significantly difference among patients with DCIS, DCIS-Mi, and IDC (P <0.001). CONCLUSIONS: Different clinicopathological, immunohistochemical, and imaging features among DCIS, DCIS-Mi, and IDC indicate that they are distinct entities. A larger sample size is needed for further study.

Predicting the factors of lateral lymph node metastasis in papillary microcarcinoma of the thyroid in eastern China.

INTRODUCTION: Lateral lymph node metastasis is common in papillary thyroid microcarcinoma (PTMC). The present study evaluated the clinicopathologic characteristics and ultrasonographic (US) findings in predicting lateral LNM from PTMC in eastern China. MATERIALS AND METHODS: A total of 176 patients with confirmed PTMC by final histological examination who underwent central lymph node dissection (LND) and lateral LND were enrolled in our study. The clinicopathological and US data from the cases were analyzed retrospectively to determine the independent predictive factors for lateral LNM. Then, a scoring system was developed on the basis of independent factors. The sum of the points for individuals was evaluated for the value in predicting lateral LNM. RESULTS: Central LNM, underlying Hashimoto's thyroiditis, upper pole location, no well-defined margin and presence of calcifications were independent predictive factors for lateral LNM on multivariate analysis. Clinicopathological and US index points were statistically significant, with ≤ 2 favoring lateral LNM negativity with a sensitivity of 83.3 %, positive predictive value of 89.6 % and negative predictive value of 72.9 %. CONCLUSIONS: When the evaluation for lateral lymph nodes from a preoperative approach is inadequate or not obvious, our scoring system for prediction of lateral LNM can be another choice. Patients with clinicopathological and US index points ≤ 2 could be considered as lateral LNM negative, so more diagnostic approach is recommended for patients with clinicopathological and US index points >2.

Preoperative BRAF mutation is predictive of occult contralateral carcinoma in patients with unilateral papillary thyroid microcarcinoma.

BACKGROUND AND OBJECTIVE: The optimal resection extent for clinically unilateral papillary thyroid microcarcinoma (PTMC) remains controversial. The objective was to investigate risk factors associated with occult contralateral carcinoma, and put emphasis on the predictive value of preoperative BRAF mutation. MATERIALS AND METHODS: 100 clinically unilateral PTMC patients all newly diagnosed, previously untreated were analyzed in a prospective cohort study. We assessed the T1799A BRAF mutation status in FNAB specimens obtained from all PTMC patients before undergoing total thyroidectomy (TT) and central lymph node dissection (CLND) for PTMC. Univariate and multivariate analyses were used to reveal the incidence of contralateral occult cancer, difference of risk factors and predictive value, with respect to the following variables: preoperative BRAF mutation status, age, gender, tumor size, multifocality of primary tumor, capsular invasion, presence of Hashimoto thyroiditis and central lymph node metastasis. RESULTS: 20 of 100 patients (20%) had occult contralateral lobe carcinoma. On multi-variate analysis, preoperative BRAF mutation (p = 0.030, OR = 3.439) and multifocality of the primary tumor (p = 0.004, OR = 9.570) were independent predictive factors for occult contralateral PTMC presence. However, there were no significant differences between the presence of occult contralateral carcinomas and age, gender, tumor size, capsular invasion, Hashimoto thyroiditis and central lymph node metastasis. CONCLUSIONS: Total thyroidectomy, including the contralateral lobe, should be considered for the treatment of unilateral PTMC if preoperative BRAF mutation is positive and/or if the observed lesion presents as a multifocal tumor in the unilateral lobe.

Factors predictive of papillary thyroid micro-carcinoma with bilateral involvement and central lymph node metastasis: a retrospective study.

BACKGROUND: The optimal resection extent for papillary thyroid microcarcinoma (PTMC) remains controversial. The objective of the study was to investigate risk factors of bilateral PTMC and central lymph node metastasis (CLNM) to guide surgical strategies for PTMC patients. METHODS: We retrospectively reviewed 211 PTMC patients who underwent total thyroidectomy (TT) and 122 clinical lymph node-negative (cN0) cases that underwent prophylactic central lymph node dissection (CLND) between 2010 and 2011. The frequency, pattern, and predictive factors for bilateral PTMC and CLNM in these patients were studied using univariate and multivariate analysis with respect to the following variables: age, gender, extrathyroidal extension (ETE), T stage, with Hashimoto thyroiditis (HT), tumor size and multifocality based on final pathology, and preoperative evaluation using ultrasonography (US). RESULTS: Fifty-four of 211 (25.6%) patients had bilateral PTMC. In multivariate analysis, multifocality (P < 0.001, OR = 23.900) and tumor size ≥7 mm (P = 0.014, OR = 2.398) based on US were independent predictive factors for bilateral PTMC which was also independently associated with multifocality (P < 0.001, OR = 29.657) and tumor size ≥7 mm (P = 0.005, OR = 2.863) based on final pathology. Among 122 cN0 patients who underwent prophylactic CLND, we found 49.2% of patients had CLNM. CLNM was independently associated with men, age <50 years and tumor size ≥7 mm based on final pathology or preoperative US. CONCLUSIONS: TT should be considered for PTMC patients who are found multifocality and tumor size ≥7 mm based on preoperative US. CLND need be considered in cN0 patients who are men, aged <50 years or tumor size ≥7 mm based on preoperative US.

Traumatic neuroma in a patient with breast cancer after mastectomy: a case report and review of the literature.

The incidence of traumatic neuroma is extremely low, especially in those patients with breast cancer after mastectomy. There are only 10 cases reported in the literature. We report a patient who developed a palpable nodular mass near the mastectomy scar. The result of excisional biopsy was traumatic neuroma. Review of the literature reveal 10 cases with breast cancer of traumatic neuromas after mastectomy. Traumatic neuroma is a benign lesion and a reparative response of the nerve to injury, either direct/indirect trauma or chronic inflammation. Benign lesions as traumatic neuromas are more rarely seen after mastectomy. However, in order to manage patients' treatment, the most critical problem is to distinguish it from recurrent breast carcinoma. Although assistant examination methods such as ultrasound and computed tomography are valuable to a certain extent, the final diagnosis can only be confirmed on pathologic examination.