Kaichiro Ikebuchi

RECQL1 and WRN Proteins Are Potential Therapeutic Targets in Head and Neck Squamous Cell Carcinoma

RECQL1 and WRN proteins are RecQ DNA helicases that participate in suppression of DNA hyper-recombination and repair. In this study, we report evidence supporting their candidacy as cancer therapeutic targets. In hypopharyngeal carcinomas, which have the worst prognosis among head and neck squamous cell carcinomas (HNSCC) that are rapidly rising in incidence, we found that RECQL1 and WRN proteins are highly expressed and that siRNA-mediated silencing of either gene suppressed carcinoma cell growth in vitro. Similarly, siRNA administration in a murine xenograft model of hypopharyngeal carcinoma markedly inhibited tumor growth. Moreover, combining either siRNA with cis-platinum (II) diammine dichloride significantly augmented the in vivo anticancer effects of this drug that is used commonly in HNSCC treatment. Notably, we observed no recurrence of some tumors following siRNA treatment in this model. Our findings offer a preclinical proof of concept for RECQL1 and WRN proteins as novel therapeutic targets to treat aggressive HNSCC and perhaps other cancers.

RB1CC1 activates the promoter and expression of RB1 in human cancer

RB1‐inducible coiled‐coil 1 (RB1CC1, also known as FIP200) is a tumor suppressor implicated in the regulation of RB1 (retinoblastoma 1) expression. However, the molecular mechanism of RB1 regulation by RB1CC1 has not been elucidated. Here, we demonstrate that nuclear RB1CC1 binds to the RB1 promoter using chromatin immunoprecipitation assays with anti‐RB1CC1 antibody. Luciferase assays with RB1 promoter reporter plasmids revealed that RB1CC1 activated the RB1 promoter through the 201 bp upstream GC‐rich region (from the initiation ATG). Electrophoretic mobility shift assay and Western blot analysis supported RB1CC1 binding to the GC‐rich region of the RB1 promoter. In addition, the C‐terminus of RB1CC1 was required for nuclear localization and subsequent RB1 promoter activation. Furthermore, the expression levels of RB1CC1 and RB1 significantly correlated with in vivo breast cancer tissues as determined by immunohistochemical analysis. These data indicate that nuclear RB1CC1 directly activates the RB1 promoter to enhance RB1 expression in cancer cells. Evaluation of RB1CC1 in various types of human cancer tissues is expected to provide useful information for clinical practice and future therapeutic strategies.

RB1CC1 activates RB1 pathway and inhibits proliferation and cologenic survival in human cancer.

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) plays a role in the enhancement of the RB1 pathway through the direct binding to a GC-rich region 201bp upstream (from the initiation ATG) of the RB1 promoter. Here, we identified hSNF5 and p53 as the binding partners of RB1CC1 by immunoprecipitation and immunofluorescence assays. Interaction between these molecules and the RB1 pathway was analyzed by the assays of chromatin immunoprecipitation, luciferase-reporter, reverse transcription-polymerase chain reaction and immunoblot. The tumor growth suppression by RB1CC1 was evaluated by flow cytometry or by a cell growth assay. The nuclear RB1CC1 complex involving hSNF5 and/or p53 activated transcription of RB1, p16 and p21, and suppressed tumor cell growth. Furthermore, nuclear RB1CC1 expression significantly correlated with those of RB1 and p16 in breast cancer tissue in vivo, and the Ki-67 proliferation index was dependent on p53 as well as RB1CC1. The present study indicates that RB1CC1 together with hSNF5 and/or p53 enhances the RB1 pathway through transcriptional activation of RB1, p16 and p21. Evaluation of RB1CC1 expression combined with RB1 and p53 status is expected to provide useful information in clinical practice and future therapeutic strategies in breast cancer.

RB1CC1 together with RB1 and p53 predicts long-term survival in Japanese breast cancer patients.

RB1-inducible coiled-coil 1 (RB1CC1) plays a significant role in the enhancement of the retinoblastoma tumor suppressor (RB1) pathway and is involved in breast cancer development. However, RB1CC1s role in clinical progression of breast cancer has not yet been evaluated, so, as a first step, it is necessary to establish its usefulness as a tool to evaluate breast cancer patients. In this report, we have analyzed the correlation between abnormalities in the RB1CC1 pathway and long-term prognosis, because disease-specific death in later periods (>5 years) of the disease is a serious problem in breast cancer. Breast cancer tissues from a large cohort in Japan were evaluated by conventional immunohistochemical methods for the presence of the molecules involved in the RB1CC1 pathway, including RB1CC1, RB1, p53, and other well-known prognostic markers for breast cancer, such as estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. The correlation between the immunohistochemical results and clinical outcomes of 323 breast cancer patients was analyzed using a Kaplan-Meier log-rank test and a multivariate Cox proportional hazards regression analysis. Absence of nuclear RB1CC1 expression was associated with the worst prognosis (Log-rank test, Chi-Square value = 17.462, p<0.0001). Dysfunction of either one of RB1CC1, RB1, or p53 was associated with the highest risk for cancer-specific death, especially related to survival lasting more than 5 years (multivariate Cox proportional hazard ratio = 3.951, 95% Confidence Interval = 1.566–9.967, p = 0.0036). Our present data demonstrate that the combined evaluation of RB1CC1, RB1 and p53 by conventional immunohistochemical analysis provides an accurate prediction of the long-term prognoses of breast cancer patients, which can be carried out as a routine clinical examination.

RB1CC1 activates the p16 promoter through the interaction with hSNF5

RB1-inducible coiled-coil 1 (RB1CC1, also known as FIP200) is involved in dephosphorylation and increase of retinoblastoma tumor suppressor protein (RB1), but the RB1CC1 molecular mechanism in the dephosphorylation of RB1 is not fully understood. We determined that RB1CC1 activates the expression of p16 (also called INK4a/CDKN2a) through the activation of its promoter, using chromatin immunoprecipitation (ChIP) and p16 promoter-luciferase reporter assays. In addition, RB1CC1 essentially requires binding with hSNF5 (also known as BAF47/INI1, a chromatin-remodeling factor) to activate the p16 promoter, in order to enhance the RB1 pathway and acts as a tumor suppressor. Evaluation of the RB1CC1 mechanism of action is expected to provide useful information for clinical practice and future therapeutic strategies in human cancers.

Prognostic significance of RB1-inducible coiled-coil 1 in salivary gland cancers

No generally agreed‐upon method is available for predicting the prognosis of salivary gland cancers. RB1‐inducible coiled‐coil 1 (RB1CC1) is a positive regulator for the retinoblastoma tumor suppressor (RB1) pathway, and is a suitable marker for evaluating the clinical course of breast cancer. We investigated whether RB1CC1 predicts the prognosis of salivary gland cancers.

Successful retrograde arterial inflow through a muscular branch in a free anterolateral thigh chimeric flap transfer

In this report, we present a case in which a free anterolateral thigh (ALT) flap was transferred for head and neck reconstruction after oropharyngeal cancer ablation, and a retrograde arterial inflow was used to salvage the flap when the main arterial pedicle showed usual repeated spasms. The flap was raised as a chimera flap comprising a fasciocutaneous flap and a vastus lateralis muscle flap. After reperfusion, the pedicle artery exhibited spasms repeatedly and vascular flow was unstable. Therefore, we performed arterial supercharge. In the distal portion of the muscle flap, a small arterial branch was dissected as a reverse‐flow arterial pedicle. The recipient artery was also a retrograde limb of the superior thyroid artery. The flap survived; however, postoperative ultrasonographic echo evaluation revealed that the spastic descending branch of the lateral circumflex femoral artery was obstructed and that the reverse‐flow muscular perforator alone nourished the whole flap. In free ALT flap transfer, a small perforator level artery was able to nourish a flap, even in a retrograde manner. Moreover, when the vasculature of the free flap is unstable, retrograde arterial supply to a small perforator can be an option to save the flap transfer.

Congenital absence of the oval window in a case of esophageal atresia

When the oval window is absent in congenital malformations of the middle ear, ossicular reconstruction can cause problems since determination of the position of the fenestra may be difficult due to lack of a reliable landmark. We present here a rare case of congenital absence of the oval window associated with esophageal atresia. Vestibular fenestration was performed and the ossicular chain was reconstructed with the malleus attachment Teflon-wire piston prosthesis. This is the first report of congenital absence of the oval window associated with esophageal atresia. A 20-year-old woman visited our hospital complaining of left hearing loss since childhood. She had congenital esophageal atresia with distal tracheoesophageal fistula, and had undergone division of tracheoesophageal fistula and esophago-esophagostomy on the day of birth. She had no history of otitis media, and although she had been examined by several otolaryngologists, the cause of her hearing loss had not been determined. On physical examination, the right auricle was normal, but the left auricle was small and generally hypoplastic by comparison (Fig 1A and B). The external auditory canals and tympanic membranes were normal. Audiograms on initial examination revealed conductive hearing loss with an air-bone gap in the left ear (Fig 2A). Tympanometry of the left ear revealed a type A pattern. The stapedial reflex was not observed in the left ear. Findings on computed tomography suggested an inadequately formed incus. Pneumatization of mastoid air cells appeared normal. Exploratory tympanotomy was performed under general anesthesia following a retroauricular incision. The long process of the incus was absent (Fig 1C). The malleus and incus

Use of systemic low-dose unfractionated heparin in microvascular head and neck reconstruction: Influence in free-flap outcomes

Abstract Background: Intravenous heparin administration is used to prevent thrombosis in free-flap transfer. However, it is unknown whether the use of heparin affects free-flap survival. The purpose of this study is to investigate the effect of heparin in free flap transfer. Methods: Two hundred and six patients who received ablative surgery for head and neck cancer were classified into three groups. Group A received ablative surgery, neck dissection, and free-flap reconstruction, and postoperatively they were administered continuous intravenous unfractionated heparin (5000–10 000 units/day) until postoperative day 7 (POD7); group B received the same procedures as group A but without heparin; group C received only ablative surgery and neck dissection without heparin. As indicators of coagulation time, the prothrombin time-international normalised ratio (PT-INR) and the activated partial thromboplastin time (APTT) were measured, before surgery and on POD1, 3, and 7. Flap failure, bleeding, haematoma formation, re-exploration, and thromboembolic events were recorded. Results: The PT-INR and APTT were 1.3–1.5-times longer in group A (p < 0.01), and 1.3-times longer (p < 0.01) in group B. The PT-INR and APTT were higher in groups A and B than C (p < 0.01). The free-flap success rate was not affected. Only the incidence of haematoma was increased in group A (p = 0.04). Conclusion: Heparin increased the haematoma formation, but did not change the incidence of free-flap failure. Thus, the intravenous low-dose heparin use does not affect microvascular flap survival.

A Case of Parapharyngeal Cyst

A rare case of a cyst in the parapharyngeal space is reported. The patient was a 30-year-old male who complained of pain on the left side of the neck. When the patient was initially examined, the posterior wall of the oropharynx was swollen. Based on CT and MRI findings, a tumor was detected in his left parapharyngeal space. It was suspected to be cystic. A fenestration was made the antenor wall of to the cyst, producing a communication between it and the oropharynx. The pathological diagnosis and treatment of parapharyngeal cyst are discussed.