Downregulation of miR-506-3p Facilitates EGFR-TKI Resistance through Induction of Sonic Hedgehog Signaling in Non-Small-Cell Lung Cancer Cell Lines.

Non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutation eventually develop resistance to EGFR-targeted tyrosine kinase inhibitors (TKIs). Treatment resistance remains the primary obstacle to the successful treatment of NSCLC. Although drug resistance mechanisms have been studied extensively in NSCLC, the regulation of these mechanisms has not been completely understood. Recently, increasing numbers of microRNAs (miRNAs) are implicated in EGFR-TKI resistance, indicating that miRNAs may serve as novel targets and may hold promise as predictive biomarkers for anti-EGFR therapy. MicroRNA-506 (miR-506) has been identified as a tumor suppressor in many cancers, including lung cancer; however, the role of miR-506 in lung cancer chemoresistance has not yet been addressed. Here we report that miR-506-3p expression was markedly reduced in erlotinib-resistant (ER) cells. We identified Sonic Hedgehog (SHH) as a novel target of miR-506-3p, aberrantly activated in ER cells. The ectopic overexpression of miR-506-3p in ER cells downregulates SHH signaling, increases E-cadherin expression, and inhibits the expression of vimentin, thus counteracting the epithelial-mesenchymal transition (EMT)-mediated chemoresistance. Our results advanced our understanding of the molecular mechanisms underlying EGFR-TKI resistance and indicated that the miR-506/SHH axis might represent a novel therapeutic target for future EGFR mutated lung cancer treatment.

Unremitting chronic skin lesions: a case of delayed diagnosis of glucagonoma.

A 54-year-old Caucasian male with history of hypertension, hyperlipidemia, insulin-dependent diabetes mellitus, and chronic skin rash of 4 years presented to the emergency department with worsening rash and weight loss. Physical examination revealed diffuse erythematous rash, skin ulceration, bullae with associated paresthesia in the lower extremities, trunk, bilateral upper extremities, and palms and soles. A computed tomography (CT) scan with contrast showed a large, heterogenously enhancing pancreatic mass measuring 9.4 × 3.8 cm with surrounding low-attenuation soft tissue thickening. Blood tests showed hemoglobin A1C of 10.0%. Glucagon level was elevated to 2,178 (normal < 80 pg/dl). Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) from the pancreatic mass was suggestive of pancreatic endocrine tumor. The tumor cells were positive for synaptophysin, chromogranin, CD56, and pan-cytokeratin with focal positivity for glucagon, suggestive of glucagonoma. The patient underwent distal pancreatectomy along with splenectomy and cholecystectomy. The glucagon level normalized to 25 pg/dl within a week of tumor resection, and during his 6-week outpatient follow up, skin rash had completely resolved.

Digging deep into diabetes: achieving better glycemic control in diabetic patients in a resident-run clinic.

Background: Diabetes mellitus (DM) affects over 30 million Americans with an estimated annual cost of $327 billion in 2017. Patients with diabetes, especially with financial and/or social hardships, pose challenges in achieving target hemoglobin A1c (HbA1c) values. Understanding patient-specific barriers offer opportunities to improve outcomes in patient care. Objective: We aimed to improve a patient's glycemic control by reducing barriers to care. Furthermore, we evaluated the impact that a resident quality improvement effort had on providing high value diabetic care. Methods: We performed a retrospective cohort study of patients with HbA1c >9.0% in an underserved, resident-run clinic. Patients were surveyed on their knowledge of diabetes and reported obstacles to achieve diabetic control. We then implemented a 12 -month customized, patient-directed, multi-modal, multidisciplinary intervention. Results: Ninety-four patients with HbA1c >9.0% were identified, 65 surveyed, and 51 included in the intervention phase. After the intervention phase, re-evaluation of HbA1c in a paired sample comparison showed that the average HbA1c had decreased by 1.41% (11.28% vs. 9.87%, p < 0.01). Among the patients included in the intervention group, approximately 8% had their HbA1c reduced by ≥50% from their baseline, 23% had their HbA1c reduced by ≥25% from their baseline and 49% had their HbA1c reduced by ≥10% from their baseline. Conclusions: A strategically designed, a patient-centered customized intervention can have a positive impact on a patient's diabetic control.

An educational intervention to increase awareness reduces unnecessary laboratory testing in an internal medicine resident-run clinic.

At our resident-run clinic in an underserved community, laboratory test costs in 2013 exceeded the government subsidy by $400 000. To optimize limited resources and improve patient care, an education program to reduce testing was implemented. Between November 2014 and January 2015, residents attended lectures on utilization of laboratory testing, focusing on standard practice guidelines, and analyses of unnecessary tests. Multivariate nonparametric statistical methods and subgroup analysis were used to evaluate cost reduction. There were 453 clinic visits during the intervention period and 471 visits during the control period. Lectures were independently associated with a significant laboratory cost reduction. Median laboratory cost per visit decreased from $106.00 to $74.00. Total cost in the study period decreased from $79 403 to $51 463. There were similar reductions of laboratory costs in two subgroups: age groups of <50 years and ≥50 years, new encounters, and follow-up visits . In the analysis of individual tests, the cost of TSH and Vitamin D tests had the greatest reduction ($8176 and $5088 respectively). An appropriate physician education program can reduce laboratory tests and costs. Screening tests with inadequate evidence support were reduced most, whereas those with proven benefits did not decrease significantly.

Human papillomavirus oncogenic E6 protein regulates human ß-defensin 3 (hBD3) expression via the tumor suppressor protein p53.

Human ß-defensin-3 (hBD3) is an epithelial cell-derived innate immune regulatory molecule overexpressed in oral dysplastic lesions and fosters a tumor-promoting microenvironment. Expression of hBD3 is induced by the epidermal growth factor receptor signaling pathway. Here we describe a novel pathway through which the high-risk human papillomavirus type-16 (HPV-16) oncoprotein E6 induces hBD3 expression in mucosal keratinocytes. Ablation of E6 by siRNA induces the tumor suppressor p53 and diminishes hBD3 in HPV-16 positive CaSki cervical cancer cells and UM-SCC-104 head and neck cancer cells. Malignant cells in HPV-16-associated oropharyngeal cancer overexpress hBD3. HPV-16 E6 induces hBD3 mRNA expression, peptide production and gene promoter activity in mucosal keratinocytes. Reduction of cellular levels of p53 stimulates hBD3 expression, while activation of p53 by doxorubicin inhibits its expression in primary oral keratinocytes and CaSki cells, suggesting that p53 represses hBD3 expression. A p53 binding site in the hBD3 gene promoter has been identified by using electrophoretic mobility shift assays and chromatin immunoprecipitation (ChIP). In addition, the p63 protein isoform ΔNp63α, but not TAp63, stimulated transactivation of the hBD3 gene and was co-expressed with hBD3 in head and neck cancer specimens. Therefore, high-risk HPV E6 oncoproteins may stimulate hBD3 expression in tumor cells to facilitate tumorigenesis of HPV-associated head and neck cancer.

Hepatitis B reactivation in cancer patients: role of prechemotherapy screening and antiviral prophylaxis.

Hepatitis B virus (HBV) infection is a potentially life-threatening condition that can be effectively prevented by vaccination. In the United States, more than 1.5 million people are infected with HBV, and that number continues to rise with the arrival of immigrants from HBV-endemic countries. Cancer is the second leading cause of death in the United States; 1 in 2 men and women will be diagnosed during their lifetime, and a large proportion of them will require chemotherapy. Chemotherapy-induced immunosuppression can result in HBV reactivation in asymptomatic HBV carriers or patients with resolved HBV infection, causing severe morbidity and mortality. The rate of HBV reactivation depends on several factors, including host and viral factors, and varies from 3-88%. Mortality rates in HBV reactivation range from 23-71%. However, a recent US survey showed that 20% of practicing oncologists never perform any type of HBV screening before the initiation of chemotherapy, and less than 40% perform HBV screening in patients who have high-risk factors for HBV or a history of hepatitis. Given the magnitude of this clinical problem, it is very important to increase awareness among physicians regarding this potentially life-threatening complication. In this article, we review the current understanding of the problem, discuss the existing guidelines from professional societies, and outline a management plan.

Constipation in Clostridium difficile infection.

A patient presented to our hospital with worsening shortness of breath, cough and respiratory distress that slowly worsened over 7-10 days. She had a viral-like illness with runny nose and cough for 1 week, which became productive of yellowish sputum. She was treated with antibiotic and steroid with clinical improvement. Her leucocyte count continued to increase despite discontinuation of both antibiotic and steroid. All culture results returned negative. She did not have any abdominal pain or diarrhoea. Her stool was positive for Clostridium difficile toxin assayed by PCR. A CT of abdomen showed distension of cecum and proximal colon. She was treated with intravenous metronidazole, oral and rectal vancomycin and intravenous immunoglobulin. She developed multi-organ failure and died.

Acute renal failure: a rare side effect of sunitinib therapy.

Sunitinib is an oral multiple tyrosine kinase receptor inhibitor. It is usually well tolerated but can cause fatigue, malaise, and rash. Sunitinib-induced cardiotoxicity has been well described, but nephrotoxicity is very rare. Here, we report a rare case of acute renal failure caused by sunitinib therapy in a 73-year-old Caucasian female who was enrolled in a phase II trial of sunitinib therapy for clear cell ovarian cancer. At presentation, her blood urea nitrogen (BUN) was 91 mg/dl and creatinine was 9.2 mg/dl. Sunitinib therapy was discontinued, and she was treated conservatively with intravenous fluid. Creatinine gradually returned to normal, and fatigue resolved. She was diagnosed with sunitinib-induced acute renal failure. The nephrologists and oncologists should be aware of sunitinib-induced rare nephrotoxicity, and patients should be closely monitored.

BOOP as a rare complication of gemcitabine therapy.

Gemcitabine is commonly used in combination with carboplatin in patients with advanced non-small-cell lung cancer (NSCLC). Gemcitabine has good clinical activity against NSCLC and is well tolerated by the patients. Myelosuppression is its dose-limiting toxicity. A potential side effect of gemcitabine is pulmonary toxicity. Among pulmonary toxicities, pneumonia, bronchospasm, acute respiratory distress syndrome, pleural effusion and interstitial pneumonitis are well documented, but bronchiolitis obliterans organising pneumonia (BOOP) is a rarely observed adverse effect of gemcitabine therapy. The authors report a female patient who presented with progressively worsening shortness of breath, low-grade fever and non-productive cough 10 days after completion of gemcitabine therapy for poorly differentiated invasive squamous cell carcinoma of lung with bone metastases. Histopathology of a transbronchial biopsy established the diagnosis of BOOP. Treatment with intravenous steroids resulted in prompt clinical improvement, but the patient later died of progression of her lung cancer.

Haematuria as an uncommon initial presenting symptom of metastatic squamous cell carcinoma (SCC) to kidney.

A 47-year-old female presented with a 2-week history of painless haematuria. Urine dipstick showed moderate leucocytes. Blood and urine cultures were negative and cytology was negative for malignant cells. Flexible cystoscopy was negative for any bladder pathology. An ultrasonogram of the abdomen showed a mass in the left kidney. CT showed a mass-like lesion within the left kidney suspicious for renal carcinoma, and cavitary lesions in both lungs. Biopsy of the lung showed clusters of atypical cells suspicious for squamous cell carcinoma (SCC), and left kidney lesion showed malignant cells derived from SCC. A whole body positron emission tomography/CT showed lesions in the lungs, left kidney and skeleton. Complete clinical examination, laboratory and imaging studies did not reveal any site of primary tumour in any part of the body. Haematuria is a very unusual initial presentation of metastatic tumour to kidney.

An antimicrobial peptide regulates tumor-associated macrophage trafficking via the chemokine receptor CCR2, a model for tumorigenesis.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a significant part of infiltrating inflammatory cells that are frequently correlated with progression and poor prognosis of a variety of cancers. Tumor cell-produced human beta-defensin-3 (hBD-3) has been associated with TAM trafficking in oral cancer; however, its involvement in tumor-related inflammatory processes remains largely unknown. METHODOLOGY: The relationship between hBD-3, monocyte chemoattractant protein-1 (MCP-1), TAMs, and CCR2 was examined using immunofluorescence microscopy in normal and oral carcinoma in situ biopsy specimens. The ability of hBD-3 to chemoattract host macrophages in vivo using a nude mouse model and analysis of hBD-3 on monocytic cell migration in vitro, applying a cross-desensitization strategy of CCR2 and its pharmacological inhibitor (RS102895), respectively, was also carried out. CONCLUSIONS/FINDINGS: MCP-1, the most frequently expressed tumor cell-associated chemokine, was not produced by tumor cells nor correlated with the recruitment of macrophages in oral carcinoma in situ lesions. However, hBD-3 was associated with macrophage recruitment in these lesions and hBD-3-expressing tumorigenic cells induced massive tumor infiltration of host macrophages in nude mice. HBD-3 stimulated the expression of tumor-promoting cytokines, including interleukin-1alpha (IL-1alpha), IL-6, IL-8, CCL18, and tumor necrosis factor-alpha (TNF-alpha) in macrophages derived from human peripheral blood monocytes. Monocytic cell migration in response to hBD-3 was inhibited by cross-desensitization with MCP-1 and the specific CCR2 inhibitor, RS102895, suggesting that CCR2 mediates monocyte/macrophage migration in response to hBD-3. Collectively, these results indicate that hBD-3 utilizes CCR2 to regulate monocyte/macrophage trafficking and may act as a tumor cell-produced chemoattractant to recruit TAMs. This novel mechanism is the first evidence of an hBD molecule orchestrating an in vivo outcome and demonstrates the importance of the innate immune system in the development of tumors.

Expression of human beta-defensin-2 in intratumoral vascular endothelium and in endothelial cells induced by transforming growth factor beta.

Human beta-defensin-2 (hBD-2) is a small cationic peptide originally identified from psoriatic skin lesions as an antimicrobial agent of the innate immune system. The expression of hBD-2 is believed to be induced exclusively in epithelial cells by microbial components and certain proinflammatory cytokines, such as interleukin-1 beta (IL-1 beta). In this study, we report, for the first time, that hBD-2 is expressed in vascular endothelial cells associated with oral squamous cell carcinoma (OSCC) and Kaposi's sarcoma lesions, but not in that of normal stroma. Expression of hBD-2 in vascular endothelial cells was further substantiated by in vitro experiments using cultured human umbilical vein endothelial cells (HUVECs). Transforming growth factor beta1 (TGF beta 1) and IL-1 beta, two well-known tumorigenic inflammatory mediators, induce hBD-2 transcript and peptide expression in HUVECs. However, TGF beta 1 does not stimulate hBD-2 expression in oral epithelial cells. In addition, proinflammatory cytokines and microbial reagents do not induce the expression of hBD-1 and hBD-3 in HUVECs. Since hBD-2 has been shown to modulate migration, proliferation, and tube formation of HUVECs in vitro and participate in immune cell trafficking, its expression in vascular endothelial cells located within malignant lesions may play a role in tumor angiogenesis and cancer metastasis.

Overexpression of human beta-defensin-3 in oral dysplasia: potential role in macrophage trafficking.

Human beta-defensins (hBDs) are small, cationic antimicrobial peptides produced by oral and other mucosal epithelia. More recently, hBDs have been shown to regulate adaptive immunity. In this study, we provide new information about the potential role of hBD-3 in the progression of oral cancer. In normal human oral epithelia, hBD-3 is produced by mitotically active cells in the basal layers of oral epithelium, whereas hBD-1 and -2 are coexpressed in the differentiated spinosum and granulosum layers. Interestingly, premalignant cells in carcinoma in situ lesions overexpress hBD-3, but not hBD-1 and hBD-2, correlating with specific recruitment and infiltration of macrophages. Our in vitro studies demonstrate that hBD-3 chemoattracts THP-1 monocytic cells and that epidermal growth factor (EGF) significantly induces hBD-3 expression in oral epithelial cells via mitogen-activated protein kinase (MAPK) kinase MEK1/2, p38 MAPK, protein kinase C (PKC), and phosphoinositide 3 kinase (PI3K), but not via Janus kinase (JAK) and signal transducer and activator of transcription (STATs). These results suggest that hBD-3 serves as a mitogen responsive gene in the initiation of oral cancer and may act as a motility signal to recruit tumor-associated macrophages.

Identification and characterization of a novel adhesin unique to oral fusobacteria.

Fusobacterium nucleatum is a gram-negative anaerobe that is prevalent in periodontal disease and infections of different parts of the body. The organism has remarkable adherence properties, binding to partners ranging from eukaryotic and prokaryotic cells to extracellular macromolecules. Understanding its adherence is important for understanding the pathogenesis of F. nucleatum. In this study, a novel adhesin, FadA (Fusobacterium adhesin A), was demonstrated to bind to the surface proteins of the oral mucosal KB cells. FadA is composed of 129 amino acid (aa) residues, including an 18-aa signal peptide, with calculated molecular masses of 13.6 kDa for the intact form and 12.6 kDa for the secreted form. It is highly conserved among F. nucleatum, Fusobacterium periodonticum, and Fusobacterium simiae, the three most closely related oral species, but is absent in the nonoral species, including Fusobacterium gonidiaformans, Fusobacterium mortiferum, Fusobacterium naviforme, Fusobacterium russii, and Fusobacterium ulcerans. In addition to FadA, F. nucleatum ATCC 25586 and ATCC 49256 also encode two paralogues, FN1529 and FNV2159, each sharing 31% identity with FadA. A double-crossover fadA deletion mutant, F. nucleatum 12230-US1, was constructed by utilizing a novel sonoporation procedure. The mutant had a slightly slower growth rate, yet its binding to KB and Chinese hamster ovarian cells was reduced by 70 to 80% compared to that of the wild type, indicating that FadA plays an important role in fusobacterial colonization in the host. Furthermore, due to its uniqueness to oral Fusobacterium species, fadA may be used as a marker to detect orally related fusobacteria. F. nucleatum isolated from other parts of the body may originate from the oral cavity.

Identification and characterization of a cell-wall anchored DNase gene in Clostridium perfringens.

Completion of the whole genome sequence of Clostridium perfringens strain 13 revealed the presence of an extracellular nuclease gene, cadA. Transcriptional analysis showed that the cadA gene is negatively regulated by the two-component VirR/VirS system and its secondary regulator VR-RNA. The CadA protein possesses an N-terminal signal sequence and a Gram-positive cell wall anchoring motif consisting of a sorting signal (LPXTG motif), a hydrophobic domain, and positively charged residues at the end of C-terminus. By comparing the DNase production between the wild type and the cadA mutant, and DNase activity assay with the recombinant truncated CadA protein, we confirmed that the cadA gene product is one of the DNases produced by C. perfringens.