Shichuan Zhang

Predictive markers for late cervical metastasis in stage I and II invasive squamous cell carcinoma of the oral tongue

Purpose: Patients with oral tongue carcinoma treated by intraoral excision only should be followed up carefully for cervical lymph node metastasis and salvaged immediately if found, because some patients have a more aggressive clinical course. The purpose of this study was to find useful markers for predicting late cervical metastasis in patients with stage I and II invasive squamous cell carcinoma of the oral tongue. Experimental Design: We investigated clinicopathologic factors and immunohistochemical biomarkers predicting late cervical metastasis in surgical specimens from 56 patients with T1–2N0M0 invasive squamous cell carcinoma of the oral tongue who did not undergo elective neck dissection. Histopathologic factors including tumor thickness, mode of invasion, Broders grade, total score of three different malignancy grading systems, eight other clinicopathologic parameters, and immunohistochemical expression of p53, cyclin D1, Ki-67, epidermal growth factor receptor, microvessel density, cyclooxygenase-2, MUC1, laminin-5 γ2, E-cadherin, and β-catenin were examined. All of the clinicopathologic factors and immunohistochemical expression of biomarkers were compared in terms of survival. Results: In the univariate analysis, tumor thickness (P = 0.009), Broders grade (P = 0.017), nest shape (P = 0.005), mode of invasion (P < 0.001), Anneroth score (P = 0.029), Bryne score (P < 0.001), and E-cadherin expression (P = 0.003) were correlated with late cervical metastasis. Multivariate analysis on late cervical metastasis revealed that tumor thickness >4 mm, mode of invasion grade 3 or 4, and E-cadherin expression were independent factors. Late cervical metastasis was the only prognostic factor for overall survival (P = 0.002). Conclusions: Our results indicate that patients with stage I and II invasive squamous cell carcinoma of the oral tongue with tumor thickness >4 mm, mode of invasion grade 3 or 4, and low expression of E-cadherin should be considered a high-risk group for late cervical metastasis when a wait-and-see policy for the neck is adopted.

Congenital bone marrow failure in DNA-PKcs mutant mice associated with deficiencies in DNA repair

Phosphorylation of DNA-PKcs is essential for activation of DNA damage repair and for the maintenance of tissue stem cell populations.

DNA Double-Strand Break Formation upon UV-Induced Replication Stress Activates ATM and DNA-PKcs Kinases

The phosphatidylinositol 3-kinase-like protein kinases, including ATM (ataxia-telangiectasia mutated), ATR (ataxia-telangiectasia and Rad3 related), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), are the main kinases activated following various assaults on DNA. Although ATM and DNA-PKcs kinases are activated upon DNA double-strand breaks, evidence suggests that these kinases are rapidly phosphorylated by ATR kinase upon UV irradiation; thus, these kinases may also participate in the response to replication stress. Using UV-induced replication stress, we further characterize whether ATM and DNA-PKcs kinase activities are also involved in the cellular response. Contrary to the rapid activation of the ATR-dependent pathway, ATM-dependent Chk2 and KAP-1 phosphorylations, as well as DNA-PKcs Ser2056 autophosphorylation, reach their peak level at 4 to 8 h after UV irradiation. The delayed kinetics of ATM- and DNA-PKcs-dependent phosphorylations also correlated with a surge in H2AX phosphorylation, suggesting that double-strand break formation resulting from collapse of replication forks is responsible for the activation of ATM and DNA-PKcs kinases. In addition, we observed that some phosphorylation events initiated by ATR kinase in the response to UV were mediated by ATM at a later phase of the response. Furthermore, the S-phase checkpoint after UV irradiation was defective in ATM-deficient cells. These results suggest that the late increase of ATM activity is needed to complement the decreasing ATR activity for maintaining a vigilant checkpoint regulation upon replication stress.

Low levels of insulin-like growth factor type 1 receptor expression at cancer cell membrane predict liver metastasis in Dukes' C human colorectal cancers.

Purpose: The aim of this study was to evaluate the prognostic significance of insulin-like growth factor type 1 receptor (IGF-1R) expression in Dukes’ C human colorectal cancers (CRCs). Experimental Design: Immunohistochemical staining for IGF-1R was done on formalin-fixed, paraffin-embedded specimens from 161 patients with curatively resected Dukes’ C CRC and at least 5-year follow-up periods. We investigated the association between the levels of IGF-1R expression and the clinicopathologic parameters. To evaluate the accurate prognostic value of IGF-1R expression, we investigated two patterns of recurrence-free survival (RFS) according to the mode of recurrence, the hepatic-RFS (H-RFS), and the nonhepatic-RFS (nH-RFS). The influence of the pattern of IGF-1R immunostaining (membranous or cytoplasmic) on RFS was also estimated. Results: High (diffuse staining) and low (focal staining) levels of IGF-1R expression were found in 45 (28%) and 116 (72%) specimens, respectively. The recurrence rate was significantly higher in the latter group (49 of 116) than the former group (9 of 45; P = 0.01). H-RFS was significantly longer for the former group than the latter group (P = 0.021), whereas no difference was found in nH-RFS between the two groups (P = 0.121). In multivariate analysis, the level of IGF-1R expression was an independent factor for H-RFS (P = 0.015) as were the depth of invasion and lymph vessel invasion (P = 0.006 and 0.022, respectively). Using a combination of the level of IGF-1R expression and these two factors, the prognostic value was further increased. When IGF-1R staining patterns (membranous or cytoplasmic) were compared, membrane staining of IGF-1R possessed prognostic significance. Conclusions: In Dukes’ C CRC, focal membrane expression of IGF-1R in the primary tumor can predict a high risk of recurrence, especially liver metastasis. Understanding the mechanisms involved could lead to new therapeutic approaches for advanced CRC.

Laminin 5 expression protects against anoikis at aerogenous spread and lepidic growth of human lung adenocarcinoma.

Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K‐Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5‐integrin‐FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma.

Inhibition of bone-derived insulin-like growth factors by a ligand-specific antibody suppresses the growth of human multiple myeloma in the human adult bone explanted in NOD/SCID mouse

Multiple myeloma (MM) is a fatal disease that affects plasma cells. Patients with MM have 1 or more osteolytic lesions in their bone tissues, where insulin‐like growth factors (IGFs; IGF‐I and IGF‐II) are mainly stored. The role of bone‐derived IGFs in the development of MM has not been extensively studied because reliable animal models are lacking. We established an animal model using a human MM cell line, RPMI8226, in nonobese diabetic/severe‐combined immunodeficient (NOD/SCID) mice implanted with human adult bone (HAB) fragments. Treatment with an anti‐human IGF‐neutralizing monoclonal antibody, KM1468, inhibited the IGF‐I‐stimulated phosphorylation of type‐I IGF receptors (IGF‐IR) in RPMI8226 cells and the activation of the downstream PI3‐K/Akt signaling pathway in vitro. KM1468 inhibited IGF‐I‐mediated RPMI8226 cell growth in a dose‐dependent manner. In the NOD/SCID‐HAB model, treatment with KM1468 significantly inhibited the growth of RPMI8226 cells (p < 0.02). These results indicated that the growth of MM cells was predominantly stimulated not by serum‐derived IGFs, but by bone‐derived IGFs. Furthermore, the targeting of bone‐derived IGFs, using a neutralizing antibody, may offer a new therapeutic strategy for MM.

Functional Intersection of ATM and DNA-Dependent Protein Kinase Catalytic Subunit in Coding End Joining during V(D)J Recombination

ABSTRACT V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA-PKcs deficiency leads to a nearly complete block in coding join formation, as DNA-PKcs is required to activate Artemis, the endonuclease that opens hairpin-sealed coding ends. In contrast to loss of DNA-PKcs protein, here we show that inhibition of DNA-PKcs kinase activity has no effect on coding join formation when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA-PKcs. Mutation of these threonine residues to alanine (DNA-PKcs3A) renders DNA-PKcs dependent on its intrinsic kinase activity during coding end joining, at a step downstream of opening hairpin-sealed coding ends. Thus, DNA-PKcs has critical functions in coding end joining beyond promoting Artemis endonuclease activity, and these functions can be regulated redundantly by the kinase activity of either ATM or DNA-PKcs.

Computer-Assisted Analysis of Biopsy Specimen Microvessels Predicts the Outcome of Esophageal Cancers Treated with Chemoradiotherapy

Purpose: A computer-assisted microvessel analysis system was developed to evaluate correlations between the architecture of biopsy specimen microvessels and the outcome for patients with esophageal cancer treated with chemoradiotherapy. Experimental Design: Biopsy specimens from 51 patients with esophageal cancer (T2-3, any N, M0) treated with chemoradiotherapy were immunostained with an anti-CD31 antibody and quantified using computerized image analysis. We evaluated the association of several microvessel factors with overall survival, including the ratio of total microvessel perimeter to total tumor area (TP/TA), the tumor hypoxic ratio, and the ratio of total microvessel number to total tumor area (TN/TA). Results from traditional manual microvessel density (MVD) hotspot count and computerized hotspot count were compared and the relation between hotspot MVD count and survival rate was evaluated. Results: The median follow-up time was 32 months. Both univariate and multivariate analyses revealed that computer-counted hotspot MVD and TN/TA and TP/TA ratios correlated significantly with the outcome of chemoradiotherapy. Kaplan-Meier survival curves showed that patients with high computer-counted hotspot MVDs and high TN/TA and TP/TA ratios had better overall survival rate than patients with low MVDs or ratios (P = 0.025, 0.008, and 0.031, respectively). Combining computer-counted MVD or TN/TA ratio with TP/TA ratio proved more predictive than any single factor. Two researcher-counted hotspot MVDs had no significant relation with outcome. Conclusion: Computer-assisted tumor microvessel analysis is a powerful tool in predicting the outcome for patients with esophageal cancer treated with chemoradiotherapy because intraobserver and interobserver variability is minimized.

Looping-out mechanism for resolution of replicative stress at telomeres

Repetitive DNA is prone to replication fork stalling, which can lead to genome instability. Here, we find that replication fork stalling at telomeres leads to the formation of t‐circle‐tails, a new extrachromosomal structure that consists of circular telomeric DNA with a single‐stranded tail. Structurally, the t‐circle‐tail resembles cyclized leading or lagging replication intermediates that are excised from the genome by topoisomerase II‐mediated cleavage. We also show that the DNA damage repair machinery NHEJ is required for the formation of t‐circle‐tails and for the resolution of stalled replication forks, suggesting that NHEJ, which is normally constitutively suppressed at telomeres, is activated in the context of replication stress. Inhibition of NHEJ or knockout of DNA‐PKcs impairs telomere replication, leading to multiple‐telomere sites (MTS) and telomere shortening. Collectively, our results support a “looping‐out” mechanism, in which the stalled replication fork is cut out and cyclized to form t‐circle‐tails, and broken DNA is religated. The telomere loss induced by replication stress may serve as a new factor that drives replicative senescence and cell aging.

Total microvessel perimeter per tumor area is a predictor of radiosensitivity in early-stage glottic carcinoma.

PURPOSE To confirm that total number of microvessels per tumor area (TN/TA) and total microvessel perimeter per tumor area (TP/TA) are predictors for radiosensitivity in early-stage glottic carcinoma. METHODS AND MATERIALS One hundred twenty consecutive patients with T1-2N0M0 glottic cancer who were treated with radical radiotherapy in three different hospitals in Japan were included in this study. Biopsy specimens from patients were immunostained with anti-CD31 antibody. The TN/TA and TP/TA of each sample were analyzed using a computer-assisted image analysis system. Patients were stratified into high and low groups according to the cut-off values derived from receiver operating characteristic curve analysis. Progression-free survival rates of the different groups were compared. The relationship of microvessel perimeter to vessel maturation was also discussed. RESULTS Survival analysis showed higher TP/TA to be associated significantly with longer progression-free survival, as compared with the lower-level group (p = 0.031). In multivariate analysis, both TP/TA (p = 0.037) and T stage (p < 0.001) proved to be independent predictive factors. Microvessel perimeter correlated closely with vessel maturity, which suggests that a tumor with high TP/TA contains more functional vessels and as a result contains more oxygenated and radiosensitive cells. CONCLUSION Assessment of TP/TA in biopsy specimens, using a computer-assisted analysis system, can predict the radiosensitivity of early-stage glottic carcinoma. High TP/TA may be a better indication for radiotherapy than for partial laryngectomy or laser excision.