Terry Hyslop

A cyclin D1/microRNA 17/20 regulatory feedback loop in control of breast cancer cell proliferation

Decreased expression of specific microRNAs (miRNAs) occurs in human tumors, which suggests a function for miRNAs in tumor suppression. Herein, levels of the miR-17-5p/miR-20a miRNA cluster were inversely correlated to cyclin D1 abundance in human breast tumors and cell lines. MiR-17/20 suppressed breast cancer cell proliferation and tumor colony formation by negatively regulating cyclin D1 translation via a conserved 3′ untranslated region miRNA-binding site, thereby inhibiting serum-induced S phase entry. The cell cycle effect of miR-17/20 was abrogated by cyclin D1 siRNA and in cyclin D1–deficient breast cancer cells. Mammary epithelial cell–targeted cyclin D1 expression induced miR-17-5p and miR-20a expression in vivo, and cyclin D1 bound the miR-17/20 cluster promoter regulatory region. In summary, these studies identify a novel cyclin D1/miR-17/20 regulatory feedback loop through which cyclin D1 induces miR-17-5p/miR-20a. In turn, miR-17/20 limits the proliferative function of cyclin D1, thus linking expression of a specific miRNA cluster to the regulation of oncogenesis.

Hypofractionated Stereotactic Radiation Therapy: An Effective Therapy for Recurrent High-Grade Gliomas

PURPOSE Salvage options for recurrent high-grade gliomas (HGGs) are limited by cumulative toxicity and limited efficacy despite advances in chemotherapeutic and radiotherapeutic techniques. Previous studies have reported encouraging survival results and favorable toxicity with fractionated stereotactic radiotherapy, and small studies have shown similar benefit using a shortened course of hypofractionated stereotactic radiation therapy (H-SRT). We sought to determine the efficacy and toxicity profile of H-SRT alone or in addition to repeat craniotomy or concomitant chemotherapy. PATIENTS AND METHODS Between 1994 and 2008, 147 patients with recurrent HGG were treated with H-SRT (median dose, 35 Gy in 3.5-Gy fractions). Cox regression models were used to analyze survival outcomes. Variables included age, surgery before H-SRT, time to first recurrence, reirradiation dose, inclusion of chemotherapy with H-SRT, and gross tumor volume (GTV). RESULTS Younger age (P = .001), smaller GTV (P = .025), and shorter time between diagnosis and recurrence (P = .034) were associated with improvement in survival from H-SRT. Doses of radiation > or = 35 Gy approached significance (P = .07). There was no significant benefit of surgical resection or chemotherapy in this population when analysis was controlled for other prognostic factors. CONCLUSION H-SRT was well tolerated and resulted in a median survival time of 11 months after H-SRT, independent of re-operation or concomitant chemotherapy. Patients who experienced recurrence within 6 months after initial treatment had an excellent response and should not be disqualified from H-SRT. This is the largest series to examine the efficacy and tolerability of H-SRT in recurrent HGG.

A randomized controlled trial of the impact of targeted and tailored interventions on colorectal cancer screening.

Colorectal cancer screening is underutilized. The objective of the current study was to determine whether targeted and tailored interventions can increase screening use.

p21CIP1 attenuates Ras- and c-Myc-dependent breast tumor epithelial mesenchymal transition and cancer stem cell-like gene expression in vivo

p21CIP1/WAF1 is a downstream effector of tumor suppressors and functions as a cyclin-dependent kinase inhibitor to block cellular proliferation. Breast tumors may derive from self-renewing tumor-initiating cells (BT-ICs), which contribute to tumor progression, recurrence, and therapy resistance. The role of p21CIP1 in regulating features of tumor stem cells in vivo is unknown. Herein, deletion of p21CIP1, which enhanced the rate of tumorigenesis induced by mammary-targeted Ha-Ras or c-Myc, enhanced gene expression profiles and immunohistochemical features of epithelial mesenchymal transition (EMT) and putative cancer stem cells in vivo. Silencing of p21CIP1 enhanced, and expression of p21CIP1 repressed, features of EMT in transformed immortal human MEC lines. p21CIP1 attenuated oncogene-induced BT-IC and mammosphere formation. Thus, the in vitro cell culture assays reflect the changes observed in vivo in transgenic mice. These findings establish a link between the loss of p21CIP1 and the acquisition of breast cancer EMT and stem cell properties in vivo.

Alternative splicing of the cyclin D1 proto-oncogene is regulated by the RNA-binding protein Sam68.

Human cyclin D1 is expressed as two isoforms derived by alternate RNA splicing, termed D1a and D1b, which differ for the inclusion of intron 4 in the D1b mRNA. Both isoforms are frequently upregulated in human cancers, but cyclin D1b displays relatively higher oncogenic potential. The splicing factors that regulate alternative splicing of cyclin D1b remain unknown despite the likelihood that they contribute to cyclin D1 oncogenicity. In this study, we report that Sam68, an RNA-binding protein frequently overexpressed in prostate cancer cells, enhances splicing of cyclin D1b and supports its expression in prostate cancer cells. Chromatin immunoprecipitation and RNA coimmunoprecipitation experiments showed that Sam68 is recruited to the human CCND1 gene encoding cyclin D1 and that it binds to cyclin D1 mRNA. Transient overexpression and RNAi knockdown experiments indicated that Sam68 acts to enhance endogenous expression of cyclin D1b. Minigene reporter assays showed that Sam68 directly affected alternative splicing of CCND1 message, with a preference for the A870 allele that is known to favor cyclin D1b splicing. Sam68 interacted with the proximal region of intron 4, and its binding correlated inversely with recruitment of the spliceosomal component U1-70K. Sam68-mediated splicing was modulated by signal transduction pathways that elicit phosphorylation of Sam68 and regulate its affinity for CCND1 intron 4. Notably, Sam68 expression positively correlates with levels of cyclin D1b, but not D1a, in human prostate carcinomas. Our results identify Sam68 as the first splicing factor to affect CCND1 alternative splicing in prostate cancer cells, and suggest that increased levels of Sam68 may stimulate cyclin D1b expression in human prostate cancers.

Association of GUCY2C expression in lymph nodes with time to recurrence and disease-free survival in pN0 colorectal cancer.

CONTEXT The established relationship between lymph node metastasis and prognosis in colorectal cancer suggests that recurrence in 25% of patients with lymph nodes free of tumor cells by histopathology (pN0) reflects the presence of occult metastases. Guanylyl cyclase 2C (GUCY2C) is a marker expressed by colorectal tumors that could reveal occult metastases in lymph nodes and better estimate recurrence risk. OBJECTIVE To examine the association of occult lymph node metastases detected by quantifying GUCY2C messenger RNA, using the reverse transcriptase-polymerase chain reaction, with recurrence and survival in patients with colorectal cancer. DESIGN, SETTING, AND PARTICIPANTS Prospective study of 257 patients with pN0 colorectal cancer enrolled between March 2002 and June 2007 at 9 US and Canadian centers (7 academic medical centers and 2 community hospitals) provided 2570 fresh lymph nodes measuring 5 mm or larger for histopathology and GUCY2C messenger RNA analysis. Patients were followed up for a median of 24 months (range, 2-63 months) for disease recurrence or death. MAIN OUTCOME MEASURES Time to recurrence (primary outcome) and disease-free survival (secondary outcome) relative to expression of GUCY2C in lymph nodes. RESULTS Thirty-two patients (12.5%) had lymph nodes negative for GUCY2C (pN0 [mol-]), and all but 2 remained free of disease during follow-up (recurrence rate, 6.3%; 95% confidence interval [CI], 0.8%-20.8%). Conversely, 225 patients (87.5%) had lymph nodes positive for GUCY2C (pN0 [mol+]), and 47 developed recurrent disease (20.9%; 95% CI, 15.8%-26.8%) (P = .006). Multivariate analyses revealed that GUCY2C in lymph nodes was an independent marker of prognosis. Patients who were pN0 (mol+) exhibited earlier time to recurrence (adjusted hazard ratio, 4.66; 95% CI, 1.11-19.57; P = .04) and reduced disease-free survival (adjusted hazard ratio, 3.27; 95% CI, 1.15-9.29; P = .03). CONCLUSION Expression of GUCY2C in histologically negative lymph nodes appears to be independently associated with time to recurrence and disease-free survival in patients with pN0 colorectal cancer.

Inbreeding Effects on Fertility in Humans: Evidence for Reproductive Compensation

The effects of inbreeding on prereproductive mortality have been demonstrated in many natural populations, including humans. However, little is known about the effects in inbred individuals who survive to adulthood. We have investigated the effects of inbreeding on fertility among inbred adult Hutterites and demonstrate significantly reduced fecundity among the most inbred Hutterite women, as evidenced by longer interbirth intervals (P=.024) and longer intervals to a recognized pregnancy (P=.010) but not by increased rates of fetal loss (P>.50). These data suggest the presence of recessive alleles that adversely affect fecundity among the population. In contrast, completed family sizes do not differ among the more and the less-inbred Hutterite women who were born after 1920, suggesting that reproductive compensation is occurring among the more-inbred and less-fecund women. This recent reproductive strategy would facilitate the maintenance of recessive alleles and contribute to an overall decline in fertility in the population.

A uroguanylin-GUCY2C endocrine axis regulates feeding in mice

Intestinal enteroendocrine cells are critical to central regulation of caloric consumption, since they activate hypothalamic circuits that decrease appetite and thereby restrict meal size by secreting hormones in response to nutrients in the gut. Although guanylyl cyclase and downstream cGMP are essential regulators of centrally regulated feeding behavior in invertebrates, the role of this primordial signaling mechanism in mammalian appetite regulation has eluded definition. In intestinal epithelial cells, guanylyl cyclase 2C (GUCY2C) is a transmembrane receptor that makes cGMP in response to the paracrine hormones guanylin and uroguanylin, which regulate epithelial cell dynamics along the crypt-villus axis. Here, we show that silencing of GUCY2C in mice disrupts satiation, resulting in hyperphagia and subsequent obesity and metabolic syndrome. This defined an appetite-regulating uroguanylin-GUCY2C endocrine axis, which we confirmed by showing that nutrient intake induces intestinal prouroguanylin secretion into the circulation. The prohormone signal is selectively decoded in the hypothalamus by proteolytic liberation of uroguanylin, inducing GUCY2C signaling and consequent activation of downstream anorexigenic pathways. Thus, evolutionary diversification of primitive guanylyl cyclase signaling pathways allows GUCY2C to coordinate endocrine regulation of central food acquisition pathways with paracrine control of intestinal homeostasis. Moreover, the uroguanylin-GUCY2C endocrine axis may provide a therapeutic target to control appetite, obesity, and metabolic syndrome.

Impact of obesity on perioperative morbidity and mortality after pancreaticoduodenectomy.

BACKGROUND Obesity has been implicated as a risk factor for perioperative and postoperative complications. The aim of this study was to determine the impact of obesity on morbidity and mortality in patients undergoing pancreaticoduodenectomy (PD). STUDY DESIGN Between January 2000 and July 2007, 262 patients underwent PD at Thomas Jefferson University Hospital, of whom 240 had complete data, including body mass index (BMI; calculated as kg/m(2)) for analysis. Data on BMI, preoperative parameters, operative details, and postoperative course were collected. Patients were categorized as obese (BMI >or= 30), overweight (BMI >or= 25 and < 30), or normal weight (BMI < 25). Complications were graded according to previously published scales. Other end points included length of postoperative hospital stay, blood loss, and operative duration. Analyses were performed using univariate and multivariable models. RESULTS There were 103 (42.9%) normal-weight, 71 (29.6%) overweight, and 66 (27.5%) obese patients. There were 5 perioperative deaths (2.1%), with no differences across BMI categories. A significant difference in median operative duration and blood loss between obese and normal-weight patients was identified (439 versus 362.5 minutes, p = 0.0004; 650 versus 500 mL, p = 0.0139). In addition, median length of stay was significantly longer for BMI (9.5 versus 8 days, p = 0.095). Although there were no significant differences in superficial wound infections, obese patients did have an increased rate of serious complications compared with normal-weight patients (24.2% versus 13.6%, respectively; p = 0.10). CONCLUSIONS Obese patients undergoing PD have a substantially increased blood loss and longer operative time but do not have a substantially increased length of postoperative hospital stay or rate of serious complications. These findings should be considered when assessing patients for operation and when counseling patients about operative risk, but they do not preclude obese individuals from undergoing definitive pancreatic operations.

Pretreatment FDG‐PET standardized uptake value as a prognostic factor for outcome in head and neck cancer

We studied the potential prognostic significance of pretreatment 18‐fluorodeoxyglucose‐positron emission tomography (FDG‐PET) standardized uptake value (SUV) in squamous cell carcinoma of the head and neck (SCCHN).