Exploration of the Correlation Between GRHL1 Expression and Tumor Microenvironment in Endometrial Cancer and Immunotherapy.

Introduction: GRHL1 belongs to the family of Grainyhead-like (GRHL). Previous studies have shown that dysregulation of growth and survival pathways is associated with the GRHL family of gene cancers. Immunotherapy with checkpoint inhibitors has changed the treatment paradigm for many tumors, including endometrial cancer (EC). However, the effect of GRHL1 on immunotherapy in EC and its relationship with immune cell infiltration are poorly understood. Methods: Differential expression of GRHL1 between EC and normal EC tissues was analyzed by searching the TCGA database, and the results were verified utilizing immunohistochemistry analyses. Next, the relationship between GRHL1, CD8+ T cells and tumor microenvironment (TME) was also investigated, and the effect of GRHL1 expression on immunotherapy in EC was evaluated. Results: According to the findings, EC tissues had elevated expression levels of GRHL1 relative to normal tissues. Patients with EC who expressed GRHL1 at high levels experienced worse overall survival (OS) and Progression-free survival (PFS) than those whose expression was lower. In addition, GRHL1 expression was negatively correlated with CD8+ T cells, and patients with high GRHL1 expression were less effective in receiving immunotherapy. Conclusion: The expression of GRHL1 was high in EC patients, and high expression of GRHL1 inhibits the proliferation of CD8+ T cells in the tumor microenvironment of EC and affect the efficacy of immunotherapy.

Receptor-interacting protein kinase 1 (RIPK1) regulates cervical cancer cells via NF-κB-TNF-α pathway: An in vitro study.

INTRODUCTION: Cervical cancer (CC) is associated with high morbidity and mortality rates in women. Members of the receptor-interacting protein kinase (RIPK) family are important regulators of inflammation and cell death. However, the characteristics, molecular functions, and expression mechanisms of RIPK1 in CC remain unclear. MATERIAL AND METHODS: To determine whether RIPK1 can be used for targeted therapy of CC, we assessed the clinical importance, biological function, and potential impact of RIPK1 in CC in 50 patients with CC. We utilized immunohistochemical staining, transfection, western blotting, cell counting kit-8 assay, colony formation assay, and wound healing assays among others, to elucidate the role of RIPK1 in CC. RESULTS: RIPK1 expression was higher in tumor tissues than in paracancerous tissues. Poor prognosis of CC was linked to RIPK1 upregulation. Furthermore, silencing RIPK1 significantly inhibited the proliferation, migration, and invasion of CC cells in vitro. We also established that RIPK1 increased cell migration, invasion, and multiplication by regulating nuclear factor kappa-B (NF-κB) and tumor necrosis factor (TNF). DISCUSSION: RIPK1 activates NF-κB and regulates TNF release to enhance the proliferation and spread of CC cells while suppressing their apoptosis. Therefore, RIPK1 plays a key role in the formation and progression of CC and is a potential target for CC treatment.

Clinicopathological and prognosis significance of RIPK1 in patients with cervical squamous cell carcinoma: a retrospective cohort study.

Background: Cervical cancer is one of the most common types of carcinoma in women and has high morbidity and mortality rates worldwide. Recurrent and metastatic disease remains difficult to treat. Receptor interacting protein kinase 1 (RIPK1) is a key molecule in mediating apoptosis, necroptosis, and inflammatory pathways downstream of death receptors and pattern recognition receptors. This study sought to explore the clinicopathological and prognosis significance of RIPK1 expression in cervical squamous cell carcinoma (CSCC). Methods: The data of 100 CSCC patients who underwent curative surgery from 2019 to 2020 were retrospectively included in this study. We collected the clinicopathological information of the patients and detected RIPK1 protein expression using immunohistochemistry. The Chi-square test and a 1-way analysis of variance were used to make comparisons between groups stratified by RIPK1 expression. A Pearson linear correlation analysis was used to evaluate the association between RIPK1 expression and the clinicopathological characteristics of the patients. A Cox regression analysis and Kaplan-Meier curves were used to analyze overall survival (OS) and progression-free survival (PFS). A multivariable regression analysis was conducted to identify the risk factors for an impaired prognosis in CSCC. Results: RIPK1 was found to be overexpressed in the CSCC tissues. RIPK1 expression was significantly associated with age, the preoperative serum squamous cell carcinoma antigen (SCC-Ag) level, lymph node metastasis, invasion depth, International Federation of Gynecology and Obstetrics (FIGO) stage, tumor size, PFS, and OS (P<0.05). The PFS and OS differed significantly among patients with RIPK1 expression (P<0.05). The multivariate analysis showed that RIPK1 was not independent risk factors for PFS and OS in CSCC patients (P>0.05). Conclusions: The expression of RIPK1 was significantly upregulated in CSCC and was associated with the clinicopathological features of CSCC. RIPK1 might serve as a novel marker that can be used to predict the prognosis of CSCC patients and as a biological target for the treatment of CSCC.

Endometrial cancer prognosis prediction using correlation models based on CDK family genes.

Cyclin-dependent kinases (CDKs) play an important role in cell division. Given that abnormal cell proliferation caused by dysregulation of cell division is one of the major causes of endometrial cancer (EC), it is important to elucidate the role of CDK family genes in the diagnosis and prognosis of EC. In this study, The Cancer Genome Atlas (TCGA) database was used to analyze the frequency of copy number variations and somatic mutations in 26 CDK family genes. Subsequently, the expression of these genes in EC was assessed, and their relationship with overall survival (OS) was examined via Kaplan-Meier analysis to assess their prognostic significance. A prognostic model based on seven CDK genes was constructed using Lasso and Cox regression, and the predictive performance of the model was analyzed using Kaplan-Meier analysis and column line plots. The correlation between CDK genes and immune cells was also examined. Patients with EC in the high-risk group had a poorer prognosis. The results of qRT-PCR and immunohistochemical analyses validated that CDK16 is highly expressed in EC tissues. Patients with EC with high CDK16 expression had worse 10-year OS than patients with low CDK16 expression. These findings suggest that the prognostic model constructed based on CDK genes can help to develop individualized and targeted treatment strategies for patients with EC.

Erb­B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53­responsive microRNA­3184­5p in cervical cancer cells.

The oncogenic role of Erb­B2 Receptor Tyrosine Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of action in cervical cancer cells. The present study employed a multipronged approach to investigate the role of ERBB2 in cervical cancer. ERBB2 and microRNA (miR)­3184­5p expression was assessed in patient­derived cervical cancer biopsy tissues, revealing that higher levels of ERBB2 and lower levels of miR­3184­5p were associated with clinicopathological indicators of cervical cancer progression. Furthermore, ERBB2 stimulated proliferation, migration and sphere­formation of cervical cancer cells in vitro. This effect was mediated by enhanced phosphatidylinositol­4,5­bisphosphate 3­kinase catalytic subunit α activity. Additionally, it was revealed that miR­3184­5p directly suppressed ERBB2 in cervical cancer cells. The p53 activator Mithramycin A stimulated p53 and miR­3184­5p expression, thereby lowering the levels of ERBB2 and attenuating proliferation, migration and sphere­formation of cervical cancer cells. In conclusion, the findings of the present study suggested ERBB2 as an oncogenic protein that may promote invasiveness in cervical cancer cells. Treatment of cervical cancer cells with the p53 activator Mithramycin A restored the levels of the endogenous ERBB2 inhibitor miR­3184­5p and may represent a novel treatment strategy for cervical cancer.

RPGRIP1L helps to establish the ciliary gate for entry of proteins.

Mutations in transition zone genes change the composition of the ciliary proteome. We isolated new mutations in RPGRIP1L (denotated as RPG1 in algae) that affect the localization of the transition zone protein NPHP4 in the model organism Chlamydomonas reinhardtii NPHP4 localization is not affected in multiple new intraflagellar transport (IFT) mutants. We compared the proteome of cilia from wild-type and mutants that affect the transition zone (RPGRIP1L) or IFT (IFT172 and DHC1b) by mass spectrometry. The rpg1-1 mutant cilia show the most dramatic increase in cytoplasmic proteins. These nonciliary proteins function in translation, membrane remodeling, ATP production and as chaperonins. These proteins are excluded in isolated cilia from fla11-1 (IFT172) and fla24-1 (DHC1b). Our data support the idea that RPGRIP1L, but not IFT proteins, acts as part of the gate for cytoplasmic proteins. The rpg1-1 cilia lack only a few proteins, which suggests that RPGRIP1L only has a minor role of in the retention of ciliary proteins. The fla11-1 mutant shows the greatest loss/reduction of proteins, and one-third of these proteins have a transmembrane domain. Hence, IFT172 may play a role in the retention of proteins.

The clinical significance of secreted protein acidic and rich in cysteine expression in breast cancer tissue and its association with prognosis.

OBJECTIVE: The aim of this study was to evaluate the secreted protein acidic and rich in cysteine (SPARC) expression in breast cancer tissue and its association with patients' clinical pathology characteristics and prognosis. MATERIALS AND METHODS: Eight-eight cases with confirmed diagnosis of breast cancer who received operation from January 2010 to February 2016 were included in this study. The SPARC expression in cancer tissue was examined by immunohistochemical method. The SPARC expression status, clinical pathology characteristics, and prognosis of included patients were recorded and evaluated. RESULTS: SPARC protein was mainly expressed in cytoplasm and stroma of tumor tissue with dark brown and purple stain. The SPARC protein-positive expression rate was 69.3% (61/88) in cancer tissue. The positive expression of SPARC in breast tissues was not significantly correlated with age, menstruation status, tumor, node, and metastasis stage, tumor size, progesterone level, and HER-2 status (P > 0.05). However, SPARC protein-positive expression was correlated with tumor differentiation (P < 0.05), estrogen receptor expression (P < 0.05), and lymph node metastasis (P < 0.05). The 3-year disease-free survival (DFS) was 60.8% and 71.2% for SPARC-positive and -negative groups with no statistical difference (P > 0.05); there was no statistical difference of disease progression risk between the SPARC-positive and -negative groups (hazard ratio = 1.78, 95% confidence interval: 0.80-3.57, P > 0.05). However, SPARC-positive and -negative patients have shown a trend of DFS difference. CONCLUSION: SPARC is closely related to the development of breast cancer and can be used as a tumor marker for breast cancer recurrence.

Serum expression level of squamous cell carcinoma antigen, highly sensitive C-reactive protein, and CA-125 as potential biomarkers for recurrence of cervical cancer.

OBJECTIVE: The aim of this study was to evaluate the serum expression levels of squamous cell carcinoma antigen (SCC-Ag), highly sensitive C-reactive protein (hs-CRP), and CA-125 as potential serum biomarkers for recurrence of cervical cancer. METHODS: Eighty-six cervical cancer patients who received radical treatment were retrospectively included in this study from February 2011 to January 2014. Of the included 86 cases, 23 were recurred within the 36 months (recurrence group [RG]) and other 63 patients did not (non-RG [NRG]). The serum levels of SCC-Ag, hs-CRP, and CA-125 were examined and compared between the two groups. The prediction recurrence sensitivity, specificity area under the receiver operating characteristic curve were calculated by STATA11.0 software (http://www.stata.com). The correlation among SCC-Ag, hs-CRP, and CA-125 were analyzed by Pearson correlation test. RESULTS: The serum levels of SCC-Ag, hs-CRP, and CA-125 were 1.29 (0.21-33.20) mg/mL, 4.78 (0.22-175.20) mg/mL, and 11.56 (2.028-123.66) IU/mL for NRG and 5.64 (0.50-136.80) mg/mL, 22.41 (0.56-588.90) mg/mL, and 25.41 (3.658-3687.00) IU/mL for RG, respectively. The serum levels of SCC-Ag, hs-CRP, and CA-125 in NG group were significant higher than those of NRG group (P < 0.05). The recurrence prediction sensitivity was 0.74, 0.65, and 0.74; specificity was 0.65, 0.63, and 0.58; area under the curve was 0.75, 0.66, and 0.67, respectively, for serum SCC-Ag, hs-CRP, and CA-125. Significant positive correlation between SCC-Ag and hs-CRP (rpearson = 0.20, P = 0.04), SCC-Ag and CA-125 (rpearson = 0.64, P < 0.001), hs-CRP and CA-125 (rpearson= -0.13, P = 0.56) was found in the RG patients. CONCLUSION: Serum SCC-Ag, hs-CRP, and CA-125 were higher in recurrence cervical patients which could be potential biomarkers for predicting cervical cancer recurrence risk.

HSP70 inhibitor combined with cisplatin suppresses the cervical cancer proliferation in vitro and transplanted tumor growth: An experimental study.

OBJECTIVE: To study the regulating effect of HSP70 inhibitor (PES) combined with cisplatin on cervical cancer proliferation in vitro and transplanted tumor growth. METHODS: Cervical cancer Hela cell lines were cultured and divided into control group, cisplatin group, PES group and cisplatin + PES group that were treated with serum-free DMEM, cisplatin with final concentration of 10 µmol/L, PES 20 µmol/L and cisplatin 10 µmol/L combined with PES with 20 µmol/L, respectively; animal models with cervical cancer xenografts were established and divided into control group, cisplatin group, PES group and cisplatin + PES group who received intra-tumor injection of normal saline, 10 µmol/L cisplatin, 20 µmol/L PES as well as 10 µmol/L cisplatin + 20 µmol/L PES, respectively. Cell proliferation activity, transplanted tumor volume and mitochondria apoptosis molecule expression were detected. RESULTS: Cell viability value and Bcl-2 mRNA expression in cells of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in cells were significantly higher than those of control group; transplanted tumor volume and the Bcl-2 mRNA expression in transplanted tumor tissue of cisplatin group, PES group and cisplatin + PES group were significantly lower than those of control group while Bax, Caspase-3 and Caspase-9 mRNA expression in transplanted tumor tissue were significantly higher than those of control group. CONCLUSIONS: HSP70 inhibitor combined with cisplatin can inhibit cervical cancer cell proliferation in vitro and transplanted tumor growth through mitochondrial apoptosis pathway.

[Inhibiting HSP70 expression enhances cisplatin sensitivity of cervical cancer cells].

OBJECTIVE: To investigate the relationship between sensitivity to cisplatin (DDP) and the expression of HSP70 in cervical cancer cells in vitro. METHODS: Cervical cancer Hela229 cells treated with different concentrations of DDP and the HSP70 inhibitor (PFT-µ) were examined for cell viability using MTT assay and colony forming ability. The cell apoptosis was analyzed by flow cytometry with propidium iodide staining and DAPI staining, and JC-1 staining was used to determine mitochondrial membrane potential. The expressions of HSP70, Bcl-2, Bax and caspase-3 were measured with Western blotting. A nude mouse model bearing Hela229 cell xenograft was used to evaluate the effect of DDP and PFT-µ on tumor growth. RESULTS: Hela229 cells expressed a higher level of HSP70 than normal cervical cells. The combined use of PFT-µ significantly enhanced the inhibitory effect of DDP (P<0.01) and increased the cell apoptosis in Hela229 cells. JC-1 staining demonstrated that DDP combined with PFT-µ more obviously reduced mitochondrial membrane potential. DDP combined with PFT-µ more strongly lowered Bcl-2 expression and increased the expressions of casepase-3 and Bax than DDP alone. In the nude mouse model, PFT-µ significantly enhanced DDP sensitivity of Hela229 cell xenografts (P<0.01). CONCLUSIONS: Inhibition of HSP70 expression can enhance the sensitivity of cervical cancer cell to DDP both in vivo and in vitro possibly by promoting cell apoptosis, suggesting the potential of HSP70 as a new target for gene therapy of cervical cancer.

A NIMA-Related Kinase Suppresses the Flagellar Instability Associated with the Loss of Multiple Axonemal Structures.

CCDC39 and CCDC40 were first identified as causative mutations in primary ciliary dyskinesia patients; cilia from patients show disorganized microtubules, and they are missing both N-DRC and inner dynein arms proteins. In Chlamydomonas, we used immunoblots and microtubule sliding assays to show that mutants in CCDC40 (PF7) and CCDC39 (PF8) fail to assemble N-DRC, several inner dynein arms, tektin, and CCDC39. Enrichment screens for suppression of pf7; pf8 cells led to the isolation of five independent extragenic suppressors defined by four different mutations in a NIMA-related kinase, CNK11. These alleles partially rescue the flagellar length defect, but not the motility defect. The suppressor does not restore the missing N-DRC and inner dynein arm proteins. In addition, the cnk11 mutations partially suppress the short flagella phenotype of N-DRC and axonemal dynein mutants, but do not suppress the motility defects. The tpg1 mutation in TTLL9, a tubulin polyglutamylase, partially suppresses the length phenotype in the same axonemal dynein mutants. In contrast to cnk11, tpg1 does not suppress the short flagella phenotype of pf7. The polyglutamylated tubulin in the proximal region that remains in the tpg1 mutant is reduced further in the pf7; tpg1 double mutant by immunofluorescence. CCDC40, which is needed for docking multiple other axonemal complexes, is needed for tubulin polyglutamylation in the proximal end of the flagella. The CCDC39 and CCDC40 proteins are likely to be involved in recruiting another tubulin glutamylase(s) to the flagella. Another difference between cnk11-1 and tpg1 mutants is that cnk11-1 cells show a faster turnover rate of tubulin at the flagellar tip than in wild-type flagella and tpg1 flagella show a slower rate. The double mutant shows a turnover rate similar to tpg1, which suggests the faster turnover rate in cnk11-1 flagella requires polyglutamylation. Thus, we hypothesize that many short flagella mutants in Chlamydomonas have increased instability of axonemal microtubules. Both CNK11 and tubulin polyglutamylation play roles in regulating the stability of axonemal microtubules.

[Significance and implication on changes of serum squamous cell carcinoma antigen in the diagnosis of recurrence squamous cell carcinoma of cervix].

OBJECTIVE: To explore the significance of serum squamous cell carcinoma antigen (SCC-Ag) in the diagnose of recurrent squamous cell carcinoma of cervix, and seek an effective approach to monitor tumor recurrence after treatment as early as possible. METHODS: All the data were collected from 1 557 patients with cervical squamous cell carcinoma treated in the First Affiliated Hospital of Bengbu Medical College from Jan. 2004 to Dec. 2010, the data of un-controlled cases(patients whose serum level of SCC- Ag failed to decrease to normal level or patients with incomplete tumor extinction)were eliminated, there were 1 394 cases of complete remission were analyzed, the median age was 46 years( rang, 18-72 years). According to International Federation of Gynecology and Obstetrics stages (FIGO, 1994), 71 cases were in stage Ia, 360 cases stage Ib, 254 cases stage IIa, 207 cases stage IIb, 95 cases stage IIIa, 289 cases stage IIIb, 106 cases stage IVa and 12 cases stage IVb. Radical hysterectomy was performed in 685 cases with stage I-IIa, concurrent radiation and chemotherapy was performed in 709 cases with stage IIb-IV. Taking serum cut off value of SCC- Ag was 1.95 µg/L, the level ≤ 1.95 µg/L as SCC- Ag negative expression and >1.95 µg/L as SCC-Ag positive expression. The positive expression status of SCC-Ag before treatment, the relationship between the post-treatment recurrence rate after SCC-Ag becoming negative and the clinicopathological features of the patients with cervical squamous cell carcinoma were analyzed, concurrently combined with gynecological check- up, pathological and imaging examination, followed by comparative analysis with the results of monitoring. RESULTS: Among 1 394 patients with cervical squamous cell carcinoma, there were 1 169 cases with positive SCC-Ag, the positive expression rate of SCC-Ag before treatment was closely related with the clinical stages and tumor size(all P < 0.01), which was not related with pathological grade and lymphatic metastasis(all P > 0.05). The positive expression of SCC-Ag in 1 169 patients before treatment turned negative after treatment, 279 patients with recurrence whose positive expression of SCC- Ag turned negative after treatment, the recurrence rate was closely related with clinical stages, pathological grade, lymphatic metastasis and tumor size(all P < 0.01). Among 279 cases of recurrent squamous cell carcinoma of cervix, the median time of the increase in serum level of SCC-Ag was 12.4 months, and the median time of clinical or imaging examination was 19.0 months. And the recurrence time in the increase of patients' serum level of SCC- Ag occurred obviously earlier than that in clinical or radiographic manifestation (P < 0.01), the median time of recurrence of the increase in serum level of SCC-Ag was obviously earlier than that of abnormality found by clinical or imaging examination(P < 0.01). Among 279 cases with recurrent squamous cell carcinoma of cervix, the serum level of SCC-Ag >5 µg/L for patients with central recurrence was 4.2% (4/96), while in the group of pelvic wall recurrence group was 56.2% (45/80)and in distant metastasis group was 87.4% (90/103). The level of SCC- Ag >5 µg/L from the patients with pelvic wall recurrence and distant metastasis were much higher than that of patients with central recurrence (P < 0.01), and the same results were shown between distant metastasis group and pelvic wall recurrent group (P < 0.01). CONCLUSIONS: SSC-Ag expression in serum of patients with cervical squamous carcinoma is closely related with clinical stages and the tumor size. Dynamic monitoring the level of SSC-Ag could contribute to the early diagnosis for tumor recurrence, but it has some limitations for patients with central recurrence, which should deserve our attention.