Robert Calaluce

Review article: Micrometastasis in colorectal carcinoma: A review

Lymph node metastasis is the most important predictor of prognosis, after surgery, in colorectal carcinoma. The term “micrometastasis” has evolved from a morphological definition to one that is used with molecular‐based techniques. We review the literature to evaluate the significance of detecting micrometastases in colorectal carcinoma, either by morphological or molecular techniques, and address technical difficulties encountered with both. Routine use of immunohistochemistry is not recommended as most studies show little change in staging or prognosis. Radioimmunoguided surgery may prove beneficial, but problems of false positives in benign diseases need to be addressed. Immunohistochemical detection of micrometastatic deposits in bone marrow aspirates holds the most promise for clinical practice. Molecular techniques are more sensitive than immunohistochemistry, but prognostic value needs to be determined. Molecular diagnostics can also determine genetic alterations and mutations that should improve our understanding of metastatic colon cancer and staging accuracy. J. Surg. Oncol. 1998;67:194–202.

The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer

BackgroundThe discordance between steady-state levels of mRNAs and protein has been attributed to posttranscriptional control mechanisms affecting mRNA stability and translation. Traditional methods of genome wide microarray analysis, profiling steady-state levels of mRNA, may miss important mRNA targets owing to significant posttranscriptional gene regulation by RNA binding proteins (RBPs).MethodsThe ribonomic approach, utilizing RNA immunoprecipitation hybridized to microarray (RIP-Chip), provides global identification of putative endogenous mRNA targets of different RBPs. HuR is an RBP that binds to the AU-rich elements (ARE) of labile mRNAs, such as proto-oncogenes, facilitating their translation into protein. HuR has been shown to play a role in cancer progression and elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR has been described to control genes in several of the acquired capabilities of cancer and has been hypothesized to be a tumor-maintenance gene, allowing for cancers to proliferate once they are established.ResultsWe used HuR RIP-Chip as a comprehensive and systematic method to survey breast cancer target genes in both MCF-7 (estrogen receptor positive, ER+) and MDA-MB-231 (estrogen receptor negative, ER-) breast cancer cell lines. We identified unique subsets of HuR-associated mRNAs found individually or in both cell types. Two novel HuR targets, CD9 and CALM2 mRNAs, were identified and validated by quantitative RT-PCR and biotin pull-down analysis.ConclusionThis is the first report of a side-by-side genome-wide comparison of HuR-associated targets in wild type ER+ and ER- breast cancer. We found distinct, differentially expressed subsets of cancer related genes in ER+ and ER- breast cancer cell lines, and noted that the differential regulation of two cancer-related genes by HuR was contingent upon the cellular environment.

Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis

Interactions between RNA binding proteins (RBPs) and genes are not well understood, especially in regulation of angiogenesis. The RBP HuR binds to the AU-rich (ARE) regions of labile mRNAs, facilitating their translation into protein and has been hypothesized to be a tumor-maintenance gene. Elevated levels of cytoplasmic HuR directly correlate with increased invasiveness and poor prognosis for many cancers, including those of the breast. HuR controls the expression of multiple genes involved in angiogenesis including VEGFa, HIF1a, and thrombospondin 1 (TSP1). We investigated the role of HuR in estrogen receptor negative (ER-) breast cancer. MDA-MB-231 cells with higher levels of HuR have alterations in cell cycle kinetics and faster growth. Unexpectedly, HuR overexpression significantly interfered with tumor growth in orthotopic mouse models. The putative mechanism seems to be an anti-angiogenetic effect by increasing expression of TSP1 but also surprisingly, down-regulation of VEGF, a target of HuR which it normally increases. Our findings reveal that HuR may be regulating a cluster of genes involved in blood vessel formation which controls tumor angiogenesis. An approach of modulating HuR levels may overcome limitations associated with monotherapies targeting tumor vessel formation.

Endoscopic colotomy closure for natural orifice transluminal endoscopic surgery using a T-fastener prototype in comparison to conventional laparoscopic suture closure

BACKGROUND Safe and efficient endoscopic closure of a colotomy is essential for transcolonic peritoneal access or endoscopic full-thickness resection of the colon, if open or laparoscopic surgery is to be avoided. OBJECTIVE To compare the feasibility and safety of colotomy closure with the newly developed Tissue Approximation System (TAS, Ethicon Endo-Surgery, Inc.) to conventional laparoscopic suture closure. DESIGN Prospective randomized survival animal study involving 16 pigs. SETTING University hospital. INTERVENTIONS Pigs were randomized for closure of a 2- to 3-cm full-thickness colotomy with the TAS or with a conventional laparoscopic running suture. MAIN OUTCOME MEASUREMENTS Success of colotomy closure, time of colotomy closure, postoperative infection, and complication rates. RESULTS Colotomies were successfully closed in all animals. Median closure time (range) was 39.5 minutes (25-95 min) in the TAS group and 23 minutes (16-40 min) in the laparoscopic group (P = .0134). There were no postoperative infections or complications. LIMITATIONS Closure with the TAS was performed under laparoscopic vision. There was no control group without closure of the colotomy site. CONCLUSIONS Colotomies are safely closed with the TAS with comparable results to laparoscopic closure. The TAS may serve as a useful tool to close full-thickness colon defects or colotomy sites made for transluminal endoscopic procedures.

Gastrotomy creation and closure for NOTES using a gastropexy technique (with video)

BACKGROUND Safe and efficient gastrotomy creation and closure is pivotal for natural orifice transluminal endoscopic surgery (NOTES). OBJECTIVE To test a method of transgastric access and closure with commercially available devices. DESIGN An animal survival study. SETTING University hospital. PATIENTS Fifteen pigs. INTERVENTIONS By using a surgical suture passer, under endoscopic guidance, 3 percutaneous stay sutures were placed, in a triangular fashion, through the gastric wall. A gastrotomy was created with a dilation balloon, which was introduced over a guidewire through the gastric wall in the center of the 3 sutures. After performing a NOTES procedure, the gastrotomy was closed by tying the sutures. Necropsies were performed after 2 to 4 weeks. MAIN OUTCOME MEASUREMENTS Success and time of gastrotomy creation and closure, and intraoperative and postoperative complications. RESULTS Gastrotomies were successfully created and closed in all the animals. The median time to create a gastrotomy was 19 minutes (range 11-85 minutes), and the median closure time was 1 minute (range 1-45 minutes). One pig died on postoperative day 1 because of peritonitis caused by a leaking gastrotomy site that extended beyond the stay sutures. There were no other gastrotomy-related complications. All gastrotomies were well healed at the necropsy. LIMITATION No control group. CONCLUSIONS We evaluated a simple method by using the principles of the PEG technique combined with a gastropexy, which is familiar to the majority of endoscopists. Strict attention to the gastrotomy site is needed, because one leak was from the gastrotomy site that extended beyond the stay sutures.

Posttranscriptional Gene Regulation of IL-17 by the RNA-Binding Protein HuR Is Required for Initiation of Experimental Autoimmune Encephalomyelitis

IL-17 is a proinflammatory cytokine produced by activated Th17 cells and other immune cells. IL-17–producing Th17 cells are major contributors to chronic inflammatory and autoimmune diseases, such as multiple sclerosis, rheumatoid arthritis, and inflammatory bowel disease. Although the transcriptional regulation of Th17 cells is well understood, the posttranscriptional regulation of IL-17 gene expression remains unknown. The RNA-binding protein HuR positively regulates the stability of many target mRNAs via binding the AU-rich elements present in the 3′ untranslated region of many inflammatory cytokines including IL-4, IL-13, and TNF-α. However, the regulation of IL-17 expression by HuR has not been established. CD4+ Th17 cells from HuR knockout mice had decreased IL-17 steady-state mRNA and protein levels compared with wild-type Th17 cells, as well as decreases in frequency of IL-17+ cells. Moreover, we demonstrated that HuR directly binds to the IL-17 mRNA 3′ untranslated region by using RNA immunoprecipitation and biotin pulldown assays. In addition, the knockout of HuR decreased cellular proliferation of CD4+ T cells. Mice with adoptively transferred HuR KO Th17 cells had delayed initiation and reduced disease severity in the onset of experimental autoimmune encephalomyelitis compared with wild-type Th17 cells. Our results reveal a HuR-induced posttranscriptional regulatory mechanism of Th17 differentiation that influences IL-17 expression. These findings may provide novel therapeutic targets for the treatment of Th17-mediated autoimmune neuroinflammation.

Coordinate Regulation of GATA-3 and Th2 Cytokine Gene Expression by the RNA-Binding Protein HuR

The posttranscriptional mechanisms whereby RNA-binding proteins (RBPs) regulate T cell differentiation remain unclear. RBPs can coordinately regulate the expression of functionally related genes via binding to shared regulatory sequences, such as the adenylate-uridylate–rich elements (AREs) present in the 3′ untranslated region (UTR) of mRNA. The RBP HuR posttranscriptionally regulates IL-4, IL-13, and other Th2 cell-restricted transcripts. We hypothesized that the ARE-bearing GATA-3 gene, a critical regulator of Th2 polarization, is under HuR control as part of its coordinate posttranscriptional regulation of the Th2 program. We report that in parallel with stimulus-induced increase in GATA-3 mRNA and protein levels, GATA-3 mRNA half-life is increased after restimulation in the human T cell line Jurkat, in human memory and Th2 cells, and in murine Th2-skewed cells. We demonstrate by immunoprecipitation of ribonucleoprotein complexes that HuR associates with the GATA-3 endogenous transcript in human T cells and found, using biotin pulldown assay, that HuR specifically interacts with its 3′UTR. Using both loss-of-function and gain-of-function approaches in vitro and in animal models, we show that HuR is a critical mediator of stimulus-induced increase in GATA-3 mRNA and protein expression and that it positively influences GATA-3 mRNA turnover, in parallel with selective promotion of Th2 cytokine overexpression. These results suggest that HuR-driven posttranscriptional control plays a significant role in T cell development and effector function in both murine and human systems. A better understanding of HuR-mediated control of Th2 polarization may have utility in altering allergic airway inflammation in human asthmatic patients.

Conditional knockout of the RNA-binding protein HuR in CD4⁺ T cells reveals a gene dosage effect on cytokine production.

The posttranscriptional mechanisms by which RNA binding proteins (RBPs) regulate T-cell differentiation and cytokine production in vivo remain unclear. The RBP HuR binds to labile mRNAs, usually leading to increases in mRNA stability and/or translation. Previous work demonstrated that HuR binds to the mRNAs encoding the Th2 transcription factor trans-acting T-cell-specific transcription factor (GATA-3) and Th2 cytokines interleukin (IL)-4 and IL-13, thereby regulating their expression. By using a novel conditional HuR knockout (KO) mouse in which HuR is deleted in activated T cells, we show that Th2-polarized cells from heterozygous HuR conditional (OX40-Cre HuRfl/+) KO mice had decreased steady-state levels of Gata3, Il4 and Il13 mRNAs with little changes at the protein level. Surprisingly, Th2-polarized cells from homozygous HuR conditional (OX40-Cre HuRfl/fl) KO mice showed increased Il2, Il4 and Il13 mRNA and protein via different mechanisms. Specifically, Il4 was transcriptionally upregulated in HuR KO T cells, whereas Il2 and Il13 mRNA stabilities increased. Additionally, when using the standard ovalbumin model of allergic airway inflammation, HuR conditional KO mice mounted a robust inflammatory response similar to mice with wild-type HuR levels. These results reveal a complex differential posttranscriptional regulation of cytokines by HuR in which gene dosage plays an important role. These findings may have significant implications in allergies and asthma, as well as autoimmune diseases and infection.

Abstract A27: Coordinate posttranscriptional regulation of breast cancer metastasis genes by RNA binding protein HuR

Breast cancer is one of the most prevalent cancers worldwide. Distance metastasis is responsible for patient mortality. Therefore, understanding the mechanisms underlying tumor pathogenesis and metastasis is crucial for the development of novel therapies as well as preventive strategies for those who are prone to breast cancer. In contrast to transcriptional gene regulation, posttranscriptional control mechanisms of gene expression are poorly understood. Yet, many metastasis genes are regulated by RNA binding proteins (RBPs) at the levels of mRNA stability and translation. The paraneoplastic antigen, HuR, is an RBP shown to regulate multiple genes that are significantly related to breast cancer metastasis by stabilizing target mRNAs and facilitating translation into proteins. Using novel techniques developed in our lab of RNA immunoprecipitation applied to microarrays (RIP-Chip) we identified novel discrete HuR-associated mRNAs in triple negative breast cancer and estrogen receptor positive breast cancer. Many of these HuR-associated mRNA transcripts were metastasis related. We investigated the role of HuR in an aggressive triple-negative breast cancer metastatic cell line, LM2. The LM2 cells were retrovirally transduced with triple fusion reporter construct encoding thymidine kinase1, GFP and firefly luciferase to obtain in vivo and in vitro tracking capabilities. The cells were further transfected with plasmid containing HA-HuR or empty vector control to demonstrate the function of HuR in LM2. HuR over-expressing clones showed greater metastatic capability by in vitro matrigel invasion assay. Furthermore, athymic mice that were intravenously injected with HuR over-expressing LM2 cells had greater (335-fold more) tumor metastasis to the lungs than empty vector control injected group as measured by IVIS imaging. Mice injected with HuR over-expressing LM2 cells were more moribund and had greater mortality as compared with mice injected with empty vector control cells. These results suggest that HuR may play a role in breast cancer metastasis by stabilizing various pro-metastatic genes. The implications of this work are twofold. First, HuR RIP-Chip can be used to identify novel cancer relevant genes and second, interference with HuR function may ameliorate distant metastasis in breast cancer, potentially providing new therapeutic approaches for treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A27.