XIST and MUC1-C form an auto-regulatory pathway in driving cancer progression.

The long non-coding RNA X-inactive specific transcript (lncRNA XIST) and MUC1 gene are dysregulated in chronic inflammation and cancer; however, there is no known interaction of their functions. The present studies demonstrate that MUC1-C regulates XIST lncRNA levels by suppressing the RBM15/B, WTAP and METTL3/14 components of the m6A methylation complex that associate with XIST A repeats. MUC1-C also suppresses the YTHDF2-CNOT1 deadenylase complex that recognizes m6A sites and contributes to XIST decay with increases in XIST stability and expression. In support of an auto-regulatory pathway, we show that XIST regulates MUC1-C expression by promoting NF-κB-mediated activation of the MUC1 gene. Of significance, MUC1-C and XIST regulate common genes associated with inflammation and stemness, including (i) miR-21 which is upregulated across pan-cancers, and (ii) TDP-43 which associates with the XIST E repeats. Our results further demonstrate that the MUC1-C/XIST pathway (i) is regulated by TDP-43, (ii) drives stemness-associated genes, and (iii) is necessary for self-renewal capacity. These findings indicate that the MUC1-C/XIST auto-regulatory axis is of importance in cancer progression.

Identification of MUC1-C as a Target for Suppressing Progression of Head and Neck Squamous Cell Carcinomas.

The MUC1-C protein is aberrantly expressed in adenocarcinomas of epithelial barrier tissues and contributes to their progression. Less is known about involvement of MUC1-C in the pathogenesis of squamous cell carcinomas (SCC). Here, we report that the MUC1 gene is upregulated in advanced head and neck SCCs (HNSCC). Studies of HNSCC cell lines demonstrate that the MUC1-C subunit regulates expression of (i) RIG-I and MDA5 pattern recognition receptors, (ii) STAT1 and IFN regulatory factors, and (iii) downstream IFN-stimulated genes. MUC1-C integrates chronic activation of the STAT1 inflammatory pathway with induction of the ∆Np63 and SOX2 genes that are aberrantly expressed in HNSCCs. In extending those dependencies, we demonstrate that MUC1-C is necessary for NOTCH3 expression, self-renewal capacity, and tumorigenicity. The findings that MUC1 associates with ∆Np63, SOX2 and NOTCH3 expression by single-cell RNA sequencing analysis further indicate that MUC1-C drives the HNSCC stem cell state and is a target for suppressing HNSCC progression. SIGNIFICANCE: This work reports a previously unrecognized role for MUC1-C in driving STAT1-mediated chronic inflammation with the progression of HNSCC and identifies MUC1-C as a druggable target for advanced HNSCC treatment.

MUC1-C is a target of salinomycin in inducing ferroptosis of cancer stem cells.

The oncogenic MUC1-C transmembrane protein is a critical effector of the cancer stem cell (CSC) state. Addiction to MUC1-C for self-renewal in the progression of human cancers has emphasized the need for development of anti-MUC1-C agents. However, there are presently no approved small molecules for targeting MUC1-C-dependent CSCs. In screening for small molecules, we identified salinomycin (SAL), an inducer of ferroptosis, as a potent inhibitor of MUC1-C signaling. We demonstrate that SAL suppresses MUC1-C expression by disrupting a NF-κB/MUC1-C auto-inductive circuit that is necessary for ferroptosis resistance. Our results show that SAL-induced MUC1-C suppression downregulates a MUC1-C→MYC pathway that activates genes encoding (i) glutathione-disulfide reductase (GSR), and (ii) the LDL receptor related protein 8 (LRP8), which inhibit ferroptosis by generating GSH and regulating selenium levels, respectively. GSR and LRP8 contribute to the function of glutathione peroxidase 4 (GPX4), an essential negative regulator of ferroptotic cell death. We demonstrate that targeting MUC1-C genetically or with the GO-203 peptide inhibitor suppresses GPX4 expression and GPX activity in association with the induction of ferroptosis. Studies of CSCs enriched by serial passage as tumorspheres further demonstrate that the effects of SAL are mediated by downregulation of MUC1-C and thereby overcoming resistance to ferroptosis. As confirmation of these results, rescue of MUC1-C downregulation with the MUC1-C cytoplasmic domain (i) reversed the suppression of GSR, LRP8 and GPX4 expression, and (ii) attenuated the induction of ferroptosis. These findings identify SAL as a unique small molecule inhibitor of MUC1-C signaling and demonstrate that MUC1-C is an important effector of resistance to ferroptosis.

MUC1-C Is a Common Driver of Acquired Osimertinib Resistance in NSCLC.

INTRODUCTION: Osimertinib is an irreversible EGFR tyrosine kinase inhibitor approved for the first-line treatment of patients with metastatic NSCLC harboring EGFR exon 19 deletions or L858R mutations. Patients treated with osimertinib invariably develop acquired resistance by mechanisms involving additional EGFR mutations, MET amplification, and other pathways. There is no known involvement of the oncogenic MUC1-C protein in acquired osimertinib resistance. METHODS: H1975/EGFR (L858R/T790M) and patient-derived NSCLC cells with acquired osimertinib resistance were investigated for MUC1-C dependence in studies of EGFR pathway activation, clonogenicity, and self-renewal capacity. RESULTS: We reveal that MUC1-C is up-regulated in H1975 osimertinib drug-tolerant persister cells and is necessary for activation of the EGFR pathway. H1975 cells selected for stable osimertinib resistance (H1975-OR) and MGH700-2D cells isolated from a patient with acquired osimertinib resistance are found to be dependent on MUC1-C for induction of (1) phospho (p)-EGFR, p-ERK, and p-AKT, (2) EMT, and (3) the resistant phenotype. We report that MUC1-C is also required for p-EGFR, p-ERK, and p-AKT activation and self-renewal capacity in acquired osimertinib-resistant (1) MET-amplified MGH170-1D #2 cells and (2) MGH121 Res#2/EGFR (T790M/C797S) cells. Importantly, targeting MUC1-C in these diverse models reverses osimertinib resistance. In support of these results, high MUC1 mRNA and MUC1-C protein expression is associated with a poor prognosis for patients with EGFR-mutant NSCLCs. CONCLUSIONS: Our findings reveal that MUC1-C is a common effector of osimertinib resistance and is a potential target for the treatment of osimertinib-resistant NSCLCs.

MUC1-C integrates aerobic glycolysis with suppression of oxidative phosphorylation in triple-negative breast cancer stem cells.

Activation of the MUC1-C protein promotes lineage plasticity, epigenetic reprogramming, and the cancer stem cell (CSC) state. The present studies performed on enriched populations of triple-negative breast cancer (TNBC) CSCs demonstrate that MUC1-C is essential for integrating activation of glycolytic pathway genes with self-renewal and tumorigenicity. MUC1-C further integrates the glycolytic pathway with suppression of mitochondrial DNA (mtDNA) genes encoding components of mitochondrial Complexes I-V. The repression of mtDNA genes is explained by MUC1-C-mediated (i) downregulation of the mitochondrial transcription factor A (TFAM) required for mtDNA transcription and (ii) induction of the mitochondrial transcription termination factor 3 (mTERF3). In support of pathogenesis that suppresses mitochondrial ROS production, targeting MUC1-C increases (i) mtDNA gene transcription, (ii) superoxide levels, and (iii) loss of self-renewal capacity. These findings and scRNA-seq analysis of CSC subpopulations indicate that MUC1-C regulates self-renewal and redox balance by integrating activation of glycolysis with suppression of oxidative phosphorylation.

MUC1-C intersects chronic inflammation with epigenetic reprogramming by regulating the set1a compass complex in cancer progression.

Chronic inflammation promotes epigenetic reprogramming in cancer progression by pathways that remain unclear. The oncogenic MUC1-C protein is activated by the inflammatory NF-κB pathway in cancer cells. There is no known involvement of MUC1-C in regulation of the COMPASS family of H3K4 methyltransferases. We find that MUC1-C regulates (i) bulk H3K4 methylation levels, and (ii) the COMPASS SET1A/SETD1A and WDR5 genes by an NF-κB-mediated mechanism. The importance of MUC1-C in regulating the SET1A COMPASS complex is supported by the demonstration that MUC1-C and WDR5 drive expression of FOS, ATF3 and other AP-1 family members. In a feedforward loop, MUC1-C, WDR5 and AP-1 contribute to activation of genes encoding TRAF1, RELB and other effectors in the chronic NF-κB inflammatory response. We also show that MUC1-C, NF-κB, WDR5 and AP-1 are necessary for expression of the (i) KLF4 master regulator of the pluripotency network and (ii) NOTCH1 effector of stemness. In this way, MUC1-C/NF-κB complexes recruit SET1A/WDR5 and AP-1 to enhancer-like signatures in the KLF4 and NOTCH1 genes with increases in H3K4me3 levels, chromatin accessibility and transcription. These findings indicate that MUC1-C regulates the SET1A COMPASS complex and the induction of genes that integrate NF-κB-mediated chronic inflammation with cancer progression.

MUC1-C is necessary for SHP2 activation and BRAF inhibitor resistance in BRAF(V600E) mutant colorectal cancer.

Colorectal cancers (CRCs) harboring the BRAF(V600E) mutation are associated with aggressive disease and resistance to BRAF inhibitors by feedback activation of the receptor tyrosine kinase (RTK)→RAS→MAPK pathway. The oncogenic MUC1-C protein promotes progression of colitis to CRC; whereas there is no known involvement of MUC1-C in BRAF(V600E) CRCs. The present work demonstrates that MUC1 expression is significantly upregulated in BRAF(V600E) vs wild-type CRCs. We show that BRAF(V600E) CRC cells are dependent on MUC1-C for proliferation and BRAF inhibitor (BRAFi) resistance. Mechanistically, MUC1-C integrates induction of MYC in driving cell cycle progression with activation of the SHP2 phosphotyrosine phosphatase, which enhances RTK-mediated RAS→ERK signaling. We demonstrate that targeting MUC1-C genetically and pharmacologically suppresses (i) activation of MYC, (ii) induction of the NOTCH1 stemness factor, and (iii) the capacity for self-renewal. We also show that MUC1-C associates with SHP2 and is required for SHP2 activation in driving BRAFi-induced feedback of ERK signaling. In this way, targeting MUC1-C in BRAFi-resistant BRAF(V600E) CRC tumors inhibits growth and sensitizes to BRAF inhibition. These findings demonstrate that MUC1-C is a target for the treatment of BRAF(V600E) CRCs and for reversing their resistance to BRAF inhibitors by suppressing the feedback MAPK pathway.

MUC1-C is a master regulator of MICA/B NKG2D ligand and exosome secretion in human cancer cells.

BACKGROUND: The MUC1-C protein evolved in mammals to protect barrier tissues from loss of homeostasis; however, MUC1-C promotes oncogenesis in association with chronic inflammation. Aberrant expression of MUC1-C in cancers has been linked to depletion and dysfunction of T cells in the tumor microenvironment. In contrast, there is no known involvement of MUC1-C in the regulation of natural killer (NK) cell function. METHODS: Targeting MUC1-C genetically and pharmacologically in cancer cells was performed to assess effects on intracellular and cell surface expression of the MHC class I chain-related polypeptide A (MICA) and MICB ligands. The MICA/B promoters were analyzed for H3K27 and DNA methylation. Shedding of MICA/B was determined by ELISA. MUC1-C interactions with ERp5 and RAB27A were assessed by coimmunoprecipitation and direct binding studies. Exosomes were isolated for analysis of secretion. Purified NK cells were assayed for killing of cancer cell targets. RESULTS: Our studies demonstrate that MUC1-C represses expression of the MICA and MICB ligands that activate the NK group 2D receptor. We show that the inflammatory MUC1-C→NF-κB pathway drives enhancer of zeste homolog 2-mediated and DNMT-mediated methylation of the MICA and MICB promoter regions. Targeting MUC1-C genetically and pharmacologically with the GO-203 inhibitor induced intracellular and cell surface MICA/B expression but not MICA/B cleavage. Mechanistically, MUC1-C regulates the ERp5 thiol oxidoreductase that is necessary for MICA/B protease digestion and shedding. In addition, MUC1-C interacts with the RAB27A protein, which is required for exosome formation and secretion. As a result, targeting MUC1-C markedly inhibited secretion of exosomes expressing MICA/B. In concert with these results, we show that targeting MUC1-C promotes NK cell-mediated killing. CONCLUSIONS: These findings uncover pleotropic mechanisms by which MUC1-C confers evasion of cancer cells to NK cell recognition and destruction.

MUC1-C Dictates PBRM1-Mediated Chronic Induction of Interferon Signaling, DNA Damage Resistance, and Immunosuppression in Triple-Negative Breast Cancer.

The polybromo-1 (PBRM1) chromatin-targeting subunit of the SWI/SNF PBAF chromatin remodeling complex drives DNA damage resistance and immune evasion in certain cancer cells through mechanisms that remain unclear. STAT1 and IRF1 are essential effectors of type I and II IFN pathways. Here, we report that MUC1-C is necessary for PBRM1 expression and that it forms a nuclear complex with PBRM1 in triple-negative breast cancer (TNBC) cells. Analysis of global transcriptional (RNA-seq) and chromatin accessibility (ATAC-seq) profiles further demonstrated that MUC1-C and PBRM1 drive STAT1 and IRF1 expression by increasing chromatin accessibility of promoter-like signatures (PLS) on their respective genes. We also found that MUC1-C, PBRM1, and IRF1 increase the expression and chromatin accessibility on PLSs of the (i) type II IFN pathway IDO1 and WARS genes and (ii) type I IFN pathway RIG-I, MDA5, and ISG15 genes that collectively contribute to DNA damage resistance and immune evasion. In support of these results, targeting MUC1-C in wild-type BRCA TNBC cells enhanced carboplatin-induced DNA damage and the loss of self-renewal capacity. In addition, MUC1-C was necessary for DNA damage resistance, self-renewal, and tumorigenicity in olaparib-resistant BRCA1-mutant TNBC cells. Analysis of TNBC tumors corroborated that (i) MUC1 and PBRM1 are associated with decreased responsiveness to chemotherapy and (ii) MUC1-C expression is associated with the depletion of tumor-infiltrating lymphocytes (TIL). These findings demonstrate that MUC1-C activates PBRM1, and thereby chromatin remodeling of IFN-stimulated genes that promote chronic inflammation, DNA damage resistance, and immune evasion. IMPLICATIONS: MUC1-C is necessary for PBRM1-driven chromatin remodeling in chronic activation of IFN pathway genes that promote DNA damage resistance and immunosuppression.

Addiction of Merkel cell carcinoma to MUC1-C identifies a potential new target for treatment.

Merkel cell carcinoma (MCC) is an aggressive malignancy with neuroendocrine (NE) features, limited treatment options, and a lack of druggable targets. There is no reported involvement of the MUC1-C oncogenic protein in MCC progression. We show here that MUC1-C is broadly expressed in MCCs and at higher levels in Merkel cell polyomavirus (MCPyV)-positive (MCCP) relative to MCPyV-negative (MCCN) tumors. Our results further demonstrate that MUC1-C is expressed in MCCP, as well as MCCN, cell lines and regulates common sets of signaling pathways related to RNA synthesis, processing, and transport in both subtypes. Mechanistically, MUC1-C (i) interacts with MYCL, which drives MCC progression, (ii) is necessary for expression of the OCT4, SOX2, KLF4, MYC, and NANOG pluripotency factors, and (iii) induces the NEUROD1, BRN2 and ATOH1 NE lineage dictating transcription factors. We show that MUC1-C is also necessary for MCCP and MCCN cell survival by suppressing DNA replication stress, the p53 pathway, and apoptosis. In concert with these results, targeting MUC1-C genetically and pharmacologically inhibits MCC self-renewal capacity and tumorigenicity. These findings demonstrate that MCCP and MCCN cells are addicted to MUC1-C and identify MUC1-C as a potential target for MCC treatment.

Dependence on the MUC1-C Oncoprotein in Classic, Variant, and Non-neuroendocrine Small Cell Lung Cancer.

Small cell lung cancer (SCLC) is a recalcitrant malignancy defined by subtypes on the basis of differential expression of the ASCL1, NEUROD1, and POU2F3 transcription factors. The MUC1-C protein is activated in pulmonary epithelial cells by exposure to environmental carcinogens and promotes oncogenesis; however, there is no known association between MUC1-C and SCLC. We report that MUC1-C is expressed in classic neuroendocrine (NE) SCLC-A, variant NE SCLC-N and non-NE SCLC-P cells and activates the MYC pathway in these subtypes. In SCLC cells characterized by NE differentiation and DNA replication stress, we show that MUC1-C activates the MYC pathway in association with induction of E2F target genes and dysregulation of mitotic progression. Our studies further demonstrate that the MUC1-C→MYC pathway is necessary for induction of (i) NOTCH2, a marker of pulmonary NE stem cells that are the proposed cell of SCLC origin, and (ii) ASCL1 and NEUROD1. We also show that the MUC1-C→MYC→NOTCH2 network is necessary for self-renewal capacity and tumorigenicity of NE and non-NE SCLC cells. Analyses of datasets from SCLC tumors confirmed that MUC1 expression in single SCLC cells significantly associates with activation of the MYC pathway. These findings demonstrate that SCLC cells are addicted to MUC1-C and identify a potential new target for SCLC treatment. IMPLICATIONS: This work uncovers addiction of SCLC cells to MUC1-C, which is a druggable target that could provide new opportunities for advancing SCLC treatment.

Prominent response to platinum-based chemotherapy in a patient with BRCA2 mutant-neuroendocrine prostate cancer and MDM2 amplification.

INTRODUCTION: Genomic profiling provides useful information for diagnosis, treatment, and prognosis, and detection of certain defects, such as DNA repair gene aberrations or microsatellite instability, can possibly lead to optimal treatment, but this testing has not been widely used to inform prostate cancer treatment. CASE PRESENTATION: A 55-year-old man sequentially treated for prostate cancer was diagnosed as neuroendocrine prostate cancer from prostate specimens resected because of urinary retention. Subsequently, he received five cycles of platinum-based chemotherapy in total and responded well. We also performed next-generation sequencing of a sample from the prostate specimen and identified a breast cancer susceptibility gene mutation with Murine Double Minute 2 amplification and loss of heterozygosity in retinoblastoma 1. CONCLUSION: We report a neuroendocrine prostate cancer patient with Murine Double Minute 2 amplification who experienced an aggressive course and for whom platinum-based chemotherapy was effective, and one of the reasons for the good response might be the breast cancer susceptibility gene mutation.

Splenic cord capillary hemangioma with non-islet cell tumor hypoglycemia: a case report.

BACKGROUND: Splenic cord capillary hemangioma is a rare benign vascular lesion classified as a splenic hamartoma. On the other hand, non-islet cell tumor hypoglycemia (NICTH) is one of the rare causes of spontaneous hypoglycemia and is considered to be one of the paraneoplastic syndromes. To the best of our knowledge, this is the first reported case of a splenic cord capillary hemangioma with NICTH. CASE PRESENTATION: A 25-year-old male was referred to our hospital with hypoglycemia. Except for his low blood sugar, there were no abnormal findings from laboratory tests, which included an endocrinological examination. Enhanced computed tomography confirmed the presence of a solid mass measuring about 6 cm in the retroperitoneum, and a tumorectomy was performed. During this operation, it became clear that the tumor turned out to be a splenic parenchyma, and as a result, a total splenectomy was performed. Microscopically, we diagnosed this as a cord capillary hemangioma, and through immunohistochemistry, we found that some tumor cells were positive for insulin-like growth factor -II. Fortunately, the hypoglycemia-related symptoms disappeared after surgical resection was performed. The patient is still alive and well without evidence of local tumor recurrence 15 years after the operation. CONCLUSIONS: Splenic cord capillary hemangioma, one of the types of splenic hamartomas, is a very rare benign vascular lesion and might be associated with hypoglycemia thought to be NICTH.

Prognostic significance of erythrocyte protein band 4.1-like5 expression in upper urinary tract urothelial carcinoma.

OBJECTIVES: The erythrocyte protein band 4.1-like5 (EPB4.1L5) regulates E-cadherin in cancer invasion and metastasis inducing epithelial-to-mesenchymal transition. This study aimed to investigate the biological significance of EPB4.1L5 in upper urinary tract urothelial carcinoma (UTUC). METHODS: Retrospective analysis of the clinical records of 165 patients with UTUC (Ta-4N0M0) subjected to radical nephroureterectomy and immunohistochemical examination of EPB4.1L5 expression in those tissues. RESULTS: The median follow-up period was 62.2 months (interquartile range = 77.0). The score of EPB4.1L5 significantly correlated with tumor grade, pathological T stage, and lymphovascular invasion (all P<0.001). The 5-year Kaplan-Meier recurrence-free survival and cancer-specific survival rates were 54.1% and 59.5% in patients with high EPB4.1L5 expression, compared with 81.6% and 87.2%, (all P<0.001) in their counterparts. Multivariate analyses revealed that high expression of EPB4.1L5 was one of the independent prognostic factors for tumor recurrence (P = 0.022, HR = 2.40) and cancer-specific survival (P = 0.015, HR = 2.94). CONCLUSION: High EPB4.1L5 expression was related to worse clinical outcome in patients with UTUC. These results indicated that EPB4.1L5 could provide prognostic information in patients with UTUC regarding epithelial-to-mesenchymal transition.

A metastatic castration resistant prostate cancer patient with multiple bone metastases has durable biochemical and radiological response to docetaxel chemotherapy.

Docetaxel chemotherapy for metastatic castration resistant prostate cancer patients has been thought palliative because the radiological response rate is low and durable response is rare. The patient was a 64-year-old man who was diagnosed with cT3aN0M0 prostate cancer and underwent external beam radiation therapy as the initial treatment. He underwent androgen deprivation therapy and 8 cycles of docetaxel chemotherapy. His PSA level decreased and became undetectable and the disease was confirmed to be stable by radiological examination. We report a rare case that a metastatic castration resistant prostate cancer patient with multiple bone metastases has durable radiological and biochemical response.

The prognostic significance of OCT4 expression in patients with prostate cancer.

Accumulating evidence suggests that OCT4 participates in tumorigenicity and malignancy in human cancers. However, the prognostic significance of OCT4 expression in prostate cancer (PCa) or predictive significance of OCT4 in docetaxel sensitivity in castration-resistant prostate cancer (CRPC) remains unclear. The aim of this study was to assess the prognostic value of OCT4 expression in PCa. We retrospectively analyzed the clinical records and evaluated the OCT4 expression in 205 patients with PCa who underwent radical prostatectomy. We examined the change of OCT4 expression in 3 patients with CRPC who underwent transurethral resection for local progression before and after docetaxel chemotherapy. OCT4 expression was significantly associated with higher pathological T stage (P < .001). The 5-year prostate-specific antigen recurrence-free survival rate was 56.8% in patients with higher OCT4 expression and 90.6% in patients with lower OCT4 expression (P < .001). Multivariate analysis revealed that high OCT4 expression was an independent prognostic indicator of prostate-specific antigen recurrence (P < .001). Elevated strong OCT4 expression in residual CRPC cells after docetaxel chemotherapy was observed in all CRPC patients, compared with before chemotherapy in corresponding specimens. Higher OCT4 expression represents a clinically relevant predictor of patient prognosis in PCa and may be a new biomarker that will provide additional prognostic information in CRPC when treated with docetaxel.

Does pelvic lymph node dissection improve the biochemical relapse-free survival in low-risk prostate cancer patients treated by laparoscopic radical prostatectomy?

BACKGROUND AND PURPOSE: The roles and criteria for pelvic lymph node dissection (PLND) are not fully evaluated in patients with low-risk prostate cancer who are treated by laparoscopic radical prostatectomy (LRP). In this study, the outcome of PLND was assessed in terms of the biochemical relapse-free survival rates of low-risk prostate cancer patients who had undergone LRP. PATIENTS AND METHODS: Included were 286 consecutive patients who were treated with LRP without previous endocrine therapy between 2002 and 2006 at our institution. Failure rates for LRP were compared in 139 patients with low-risk prostate cancer between those who underwent PLND (n=85) and those who did not (n=54). Biochemical relapse-free survival for each group was estimated by Kaplan-Meier analysis. RESULTS: The mean number of retrieved lymph nodes was 5.4 ± 0.4 (range 2-22). The 5- and 7-year biochemical relapse-free survival rates were 90.1% and 88.3% in patients with PLND, and 82.4% and 82.4% in those without PLND (P=0.278), respectively (median follow-up 69.4 mos). None of the 85 patients undergoing PLND had positive lymph nodes. Only one patient had symptomatic lymphocele, and he was treated as an inpatient. The average time needed for PLND was 16 minutes, which corresponded to 7% of the entire operative time. CONCLUSION: These results indicate that the dissection of pelvic lymph nodes is not related to biochemical relapse-free survival. The omission of PLND in patients with low-risk prostate cancer not only does not adversely affect biochemical relapse-free survival, but might decrease the incidence of complication and operative time of LRP.