USA Mitchell Cancer Institute

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is thatthere is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure flux through the autophagy pathway (i.e., the completeprocess including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defined as increasedautophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in manycases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as forreviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multipleassays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation, it is imperative to target by gene knockout or RNA interference more than one autophagyrelated protein. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways implying that not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular assays, we hope to encourage technical innovation in the field.

The metabolic properties of cancer cells diverge significantly from those of normal cells. Energy production in cancer cells is abnormally dependent on aerobic glycolysis. In addition to the dependency on glycolysis, cancer cells have other atypical metabolic characteristics such as increased fatty acid synthesis and increased rates of glutamine metabolism. Emerging evidence shows that many features characteristic to cancer cells, such as dysregulated Warburg-like glucose metabolism, fatty acid synthesis and glutaminolysis are linked to therapeutic resistance in cancer treatment. Therefore, targeting cellular metabolism may improve the response to cancer therapeutics and the combination of chemotherapeutic drugs with cellular metabolism inhibitors may represent a promising strategy to overcome drug resistance in cancer therapy. Recently, several review articles have summarized the anticancer targets in the metabolic pathways and metabolic inhibitor-induced cell death pathways, however, the dysregulated metabolism in therapeutic resistance, which is a highly clinical relevant area in cancer metabolism research, has not been specifically addressed. From this unique angle, this review article will discuss the relationship between dysregulated cellular metabolism and cancer drug resistance and how targeting of metabolic enzymes, such as glucose transporters, hexokinase, pyruvate kinase M2, lactate dehydrogenase A, pyruvate dehydrogenase kinase, fatty acid synthase and glutaminase can enhance the efficacy of common therapeutic agents or overcome resistance to chemotherapy or radiotherapy.

Mitochondria are highly dynamic, maternally inherited cytoplasmic organelles, which fulfill cellular energy demand through the oxidative phosphorylation system. Besides, they play an active role in calcium and damage-associated molecular patterns signaling, amino acid, and lipid metabolism, and apoptosis. Thus, the maintenance of mitochondrial integrity and homeostasis is extremely critical, which is achieved through continual fusion and fission. Mitochondrial fusion allows the transfer of gene products between mitochondria for optimal functioning, especially under metabolic and environmental stress. On the other hand, fission is crucial for mitochondrial division and quality control. The imbalance between these two processes is associated with various ailments such as cancer, neurodegenerative and cardiovascular diseases. This review discusses the molecular mechanisms that control mitochondrial fusion and fission and how the disruption of mitochondrial dynamics manifests into various disease conditions.

Exosomes have received significant attention for their role in pathobiological processes and are being explored as a tool for disease diagnosis and management. Consequently, various isolation methods based on different principles have been developed for exosome isolation. Here we compared the efficacy of four kits from Invitrogen, 101Bio, Wako and iZON along with conventional ultracentrifugation-based method for exosome yield, purity and quality. Cell culture supernatant was used as an abundant source of exosomes, and exosome quantity, size-distribution, zeta-potential, marker-expression and RNA/protein quality were determined. The Invitrogen kit gave the highest yield but the preparation showed broader size-distribution likely due to microvesicle co-precipitation and had the least dispersion stability. Other preparations showed <150 nm size range and good stability. Preparation from iZON column; however, had a broader size-distribution in the lower size range suggestive of some impurities of non-vesicular aggregates. RNA quality from all preparations was comparable; however, proteins from Invitrogen method-based exosomal preparation showed polyethylene glycol (PEG) contamination in mass spectrometry. Chemical impurities from the precipitant could also be the cause of toxicity of Invitrogen method-based exosomal preparation in biological growth measurement assay. Together, these findings should serve as a guide to choose and further optimize exosome isolation methods for their desired downstream applications.

microRNAs (miRNAs) are noncoding small RNAs that regulate gene expression at the translational level by mainly interacting with 3' UTRs of their target mRNAs. Archived formalin-fixed paraffin-embedded (FFPE) specimens represent excellent resources for biomarker discovery. Currently there is a lack of systematic analysis on the stability of miRNAs and optimized conditions for expression analysis using FFPE samples. In this study, the expression of miRNAs from FFPE samples was analyzed using high-throughput locked nucleic acid-based miRNA arrays. The effect of formalin fixation on the stability of miRNAs was also investigated using miRNA real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis. The stability of miRNAs of archived colorectal cancer FFPE specimens was characterized with samples dating back up to 10 yr. Our results showed that the expression profiles of miRNAs were in good correlation between 1 mug of fresh frozen and 1-5 mug of FFPE samples (correlation coefficient R (2) = 0.86-0.89). Different formalin fixation times did not change the stability of miRNAs based on real-time qRT-PCR analysis. There are no significant differences of representative miRNA expression among 40 colorectal cancer FFPE specimens. This study provides a foundation for miRNA investigation using FFPE samples in cancer and other types of diseases.

BACKGROUND: The process of malignant transformation, progression and metastasis of melanoma is poorly understood. Gene expression profiling of human cancer has allowed for a unique insight into the genes that are involved in these processes. Thus, we have attempted to utilize this approach through the analysis of a series of primary, non-metastatic cutaneous tumors and metastatic melanoma samples. METHODS: We have utilized gene microarray analysis and a variety of molecular techniques to compare 40 metastatic melanoma (MM) samples, composed of 22 bulky, macroscopic (replaced) lymph node metastases, 16 subcutaneous and 2 distant metastases (adrenal and brain), to 42 primary cutaneous cancers, comprised of 16 melanoma, 11 squamous cell, 15 basal cell skin cancers. A Human Genome U133 Plus 2.0 array from Affymetrix, Inc. was utilized for each sample. A variety of statistical software, including the Affymetrix MAS 5.0 analysis software, was utilized to compare primary cancers to metastatic melanomas. Separate analyses were performed to directly compare only primary melanoma to metastatic melanoma samples. The expression levels of putative oncogenes and tumor suppressor genes were analyzed by semi- and real-time quantitative RT-PCR (qPCR) and Western blot analysis was performed on select genes. RESULTS: We find that primary basal cell carcinomas, squamous cell carcinomas and thin melanomas express dramatically higher levels of many genes, including SPRR1A/B, KRT16/17, CD24, LOR, GATA3, MUC15, and TMPRSS4, than metastatic melanoma. In contrast, the metastatic melanomas express higher levels of genes such as MAGE, GPR19, BCL2A1, MMP14, SOX5, BUB1, RGS20, and more. The transition from non-metastatic expression levels to metastatic expression levels occurs as melanoma tumors thicken. We further evaluated primary melanomas of varying Breslow's tumor thickness to determine that the transition in expression occurs at different thicknesses for different genes suggesting that the "transition zone" represents a critical time for the emergence of the metastatic phenotype. Several putative tumor oncogenes (SPP-1, MITF, CITED-1, GDF-15, c-Met, HOX loci) and suppressor genes (PITX-1, CST-6, PDGFRL, DSC-3, POU2F3, CLCA2, ST7L), were identified and validated by quantitative PCR as changing expression during this transition period. These are strong candidates for genes involved in the progression or suppression of the metastatic phenotype. CONCLUSION: The gene expression profiling of primary, non-metastatic cutaneous tumors and metastatic melanoma has resulted in the identification of several genes that may be centrally involved in the progression and metastatic potential of melanoma. This has very important implications as we continue to develop an improved understanding of the metastatic process, allowing us to identify specific genes for prognostic markers and possibly for targeted therapeutic approaches.

Lack of immune system cells or impairment in differentiation of immune cells is the basis for many chronic diseases. Metabolic changes could be the root cause for this immune cell impairment. These changes could be a result of altered transcription, cytokine production from surrounding cells, and changes in metabolic pathways. Immunity and mitochondria are interlinked with each other. An important feature of mitochondria is it can regulate activation, differentiation, and survival of immune cells. Additionally, it can also release signals such as mtDNA and mtROS to regulate transcription of immune cells. From current literature, we found that mitochondria can regulate immunity in different ways. Firstly, alterations in metabolic pathways (TCA cycle, OXPHOS, and FAO) and mitochondria induced transcriptional changes can lead to entirely different outcomes in immune cells. For example, M1 macrophages exhibit a broken TCA cycle and have a pro-inflammatory role. In contrast, M2 macrophages undergo β-oxidation to produce anti-inflammatory responses. Additionally, amino acid metabolism, especially arginine, glutamine, serine, glycine, and tryptophan, is critical for T cell differentiation and macrophage polarization. Secondly, mitochondria can activate the inflammatory response. For instance, MAVS signaling and NLRP3 can be activated by mitochondria. Thirdly, mitochondrial mass and mobility can be influenced by fission and fusion. Fission and fusion can influence immune functions. Lastly, mitochondria are placed near the endoplasmic reticulum (ER) in immune cells. Therefore, mitochondria and ER junction signaling can also influence immune cell metabolism. Mitochondrial machinery such as metabolic pathways, amino acid metabolism, antioxidant systems, mitochondrial dynamics, mtDNA, mitophagy, and mtROS are crucial for immune functions. Here, we have demonstrated how mitochondria coordinate to alter immune responses and how changes in mitochondrial machinery contribute to alterations in immune responses. A better understanding of the molecular components of mitochondria is necessary. This can help in the development of safe and effective immune therapy or prevention of chronic diseases. In this review, we have presented an updated prospective of the mitochondrial machinery that drives various immune responses.

Paclitaxel (Taxol) is an effective chemotherapeutic agent for treatment of cancer patients. Despite impressive initial clinical responses, the majority of patients eventually develop some degree of resistance to Taxol-based therapy. The mechanisms underlying cancer cells resistance to Taxol are not fully understood. MicroRNA (miRNA) has emerged to play important roles in tumorigenesis and drug resistance. However, the interaction between the development of Taxol resistance and miRNA has not been previously explored. In this study we utilized a miRNA array to compare the differentially expressed miRNAs in Taxol-resistant and their Taxol-sensitive parental cells. We verified that miR-125b, miR-221, miR-222, and miR-923 were up-regulated in Taxol-resistant cancer cells by real-time PCR. We further investigated the role and mechanisms of miR-125b in Taxol resistance. We found that miR-125b was up-regulated in Taxol-resistant cells, causing a marked inhibition of Taxol-induced cytotoxicity and apoptosis and a subsequent increase in the resistance to Taxol in cancer cells. Moreover, we demonstrated that the pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) is a direct target of miR-125b. Down-regulation of Bak1 suppressed Taxol-induced apoptosis and led to an increased resistance to Taxol. Restoring Bak1 expression by either miR-125b inhibitor or re-expression of Bak1 in miR-125b-overexpressing cells recovered Taxol sensitivity, overcoming miR-125-mediated Taxol resistance. Taken together, our data strongly support a central role for miR-125b in conferring Taxol resistance through the suppression of Bak1 expression. This finding has important implications in the development of targeted therapeutics for overcoming Taxol resistance in a number of different tumor histologies.

BACKGROUND: Taxol is one of the most effective chemotherapeutic agents for the treatment of patients with breast cancer. Despite impressive clinical responses initially, the majority of patients eventually develop resistance to Taxol. Lactate dehydrogenase-A (LDH-A) is one of the predominant isoforms of LDH expressed in breast tissue, which controls the conversion of pyruvate to lactate and plays an important role in glucose metabolism. In this study we investigated the role of LDH-A in mediating Taxol resistance in human breast cancer cells. RESULTS: Taxol-resistant subclones, derived from the cancer cell line MDA-MB-435, sustained continuous growth in high concentrations of Taxol while the Taxol-sensitive cells could not. The increased expression and activity of LDH-A were detected in Taxol-resistant cells when compared with their parental cells. The downregulation of LDH-A by siRNA significantly increased the sensitivity of Taxol-resistant cells to Taxol. A higher sensitivity to the specific LDH inhibitor, oxamate, was found in the Taxol-resistant cells. Furthermore, treating cells with the combination of Taxol and oxamate showed a synergistical inhibitory effect on Taxol-resistant breast cancer cells by promoting apoptosis in these cells. CONCLUSION: LDH-A plays an important role in Taxol resistance and inhibition of LDH-A re-sensitizes Taxol-resistant cells to Taxol. This supports that Warburg effect is a property of Taxol resistant cancer cells and may play an important role in the development of Taxol resistance. To our knowledge, this is the first report showing that the increased expression of LDH-A plays an important role in Taxol resistance of human breast cancer cells. This study provides valuable information for the future development and use of targeted therapies, such as oxamate, for the treatment of patients with Taxol-resistant breast cancer.

OBJECTIVE: To determine the prognostic significance of histologic type/subtype in a large series of patients with primary resected retroperitoneal sarcoma. BACKGROUND: The histologic diversity and rarity of retroperitoneal sarcoma has hampered the ability to predict patient outcome. METHODS: From a single-institution, prospective database, 675 patients treated surgically for primary, nonmetastatic retroperitoneal sarcoma during 1982 to 2010 were identified and histologic type/subtype was reviewed. Clinicopathologic variables were analyzed for association with disease-specific death (DSD), local recurrence (LR), and distant recurrence (DR). RESULTS: Median follow-up for survivors was 7.5 years. The predominant histologies were well-differentiated liposarcoma, dedifferentiated liposarcoma, and leiomyosarcoma. Five-year cumulative incidence of DSD was 31%, and factors independently associated with DSD were R2 resection, resection of 3 or more contiguous organs, and histologic type. Five-year cumulative incidence for LR was 39% and for DR was 24%. R1 resection, age, tumor size, and histologic type were independently associated with LR; size, resection of 3 or more organs, and histologic type were independently associated with DR. Liposarcoma and leiomyosarcoma were associated with late recurrence and DSD (as long as 15 years from diagnosis). For solitary fibrous tumor, LR was uncommon (<10%), but early distant recurrence was common (36% at 5 years). Nomograms were developed to predict DSD, LR, and DR. CONCLUSIONS: Histologic type/subtype is the most important independent predictor of DSD, LR, and DR in primary retroperitoneal sarcoma. Histology predicts the pattern and incidence of LR and DR and will aid in more accurate patient counseling and selection of patients for adjuvant therapy trials.

Bisphenol A (BPA) has become a target of intense public scrutiny since concerns about its association with human diseases such as obesity, diabetes, reproductive disorders, and cancer have emerged. BPA is a highly prevalent chemical in consumer products, and human exposure is thought to be ubiquitous. Numerous studies have demonstrated its endocrine disrupting properties and attributed exposure with cytotoxic, genotoxic, and carcinogenic effects; however, the results of these studies are still highly debated and a consensus about BPA's safety and its role in human disease has not been reached. One of the contributing factors is a lack of molecular mechanisms or modes of action that explain the diverse and pleiotropic effects observed after BPA exposure. The increase in BPA research seen over the last ten years has resulted in more studies that examine molecular mechanisms and revealed links between BPA-induced oxidative stress and human disease. Here, a review of the current literature examining BPA exposure and the induction of reactive oxygen species (ROS) or oxidative stress will be provided to examine the landscape of the current BPA literature and provide a framework for understanding how induction of oxidative stress by BPA may contribute to the pleiotropic effects observed after exposure. Environ. Mol. Mutagen. 58:60-71, 2017. © 2017 Wiley Periodicals, Inc.

UNLABELLED: Patients with pancreatic and biliary carcinomas lack personalized treatment options, in part because biopsies are often inadequate for molecular characterization. Cell-free DNA (cfDNA) sequencing may enable a precision oncology approach in this setting. We attempted to prospectively analyze 54 genes in tumor and cfDNA for 26 patients. Tumor sequencing failed in 9 patients (35%). In the remaining 17, 90.3% (95% confidence interval, 73.1%-97.5%) of mutations detected in tumor biopsies were also detected in cfDNA. The diagnostic accuracy of cfDNA sequencing was 97.7%, with 92.3% average sensitivity and 100% specificity across five informative genes. Changes in cfDNA correlated well with tumor marker dynamics in serial sampling (r = 0.93). We demonstrate that cfDNA sequencing is feasible, accurate, and sensitive in identifying tumor-derived mutations without prior knowledge of tumor genotype or the abundance of circulating tumor DNA. cfDNA sequencing should be considered in pancreatobiliary cancer trials where tissue sampling is unsafe, infeasible, or otherwise unsuccessful. SIGNIFICANCE: Precision medicine efforts in biliary and pancreatic cancers have been frustrated by difficulties in obtaining adequate tumor tissue for next-generation sequencing. cfDNA sequencing reliably and accurately detects tumor-derived mutations, paving the way for precision oncology approaches in these deadly diseases.

BACKGROUND: Chemoresistance is a significant clinical problem in pancreatic cancer (PC) and underlying molecular mechanisms still remain to be completely understood. Here we report a novel exosome-mediated mechanism of drug-induced acquired chemoresistance in PC cells. METHODS: Differential ultracentrifugation was performed to isolate extracellular vesicles (EVs) based on their size from vehicle- or gemcitabine-treated PC cells. Extracellular vesicles size and subtypes were determined by dynamic light scattering and marker profiling, respectively. Gene expression was examined by qRT-PCR and/or immunoblot analyses, and direct targeting of DCK by miR-155 was confirmed by dual-luciferase 3'-UTR reporter assay. Flow cytometry was performed to examine the apoptosis indices and reactive oxygen species (ROS) levels in PC cells using specific dyes. Cell viability was determined using the WST-1 assay. RESULTS: Conditioned media (CM) from gemcitabine-treated PC cells (Gem-CM) provided significant chemoprotection to subsequent gemcitabine toxicity and most of the chemoresistance conferred by Gem-CM resulted from its EVs fraction. Sub-fractionation grouped EVs into distinct subtypes based on size distribution and marker profiles, and exosome (Gem-Exo) was the only sub-fraction that imparted chemoresistance. Gene expression analyses demonstrated upregulation of SOD2 and CAT (ROS-detoxifying genes), and downregulation of DCK (gemcitabine-metabolising gene) in Gem-Exo-treated cells. SOD/CAT upregulation resulted, at least in part, from exosome-mediated transfer of their transcripts and they suppressed basal and gemcitabine-induced ROS production, and partly promoted chemoresistance. DCK downregulation occurred through exosome-delivered miR-155 and either the functional suppression of miR-155 or restoration of DCK led to marked abrogation of Gem-Exo-mediated chemoresistance. CONCLUSIONS: Together, these findings establish a novel role of exosomes in mediating the acquired chemoresistance of PC.

// Yaojie Fu 1, 2, 3 , Shanshan Liu 1, 2, 3 , Shanghelin Yin 1, 2, 3 , Weihong Niu 1, 2 , Wei Xiong 1, 2 , Ming Tan 4 , Guiyuan Li 1, 2 and Ming Zhou 1, 2 1 The Key Laboratory of Carcinogenesis of The Chinese Ministry of Health, Xiangya Hospital, Central South University, Changsha, Hunan 410078, P. R. China 2 Cancer Research Institute, Central South University, Changsha, Hunan 410078, P. R. China 3 Medical School of Xiangya, Central South University, Changsha, Hunan 410013, P. R. China 4 Mitchell Cancer Institute, University of South Alabama, Mobile, AL 36604, USA Correspondence to: Ming Zhou, email: zhouming2001@163.com Keywords: the Warburg effect, the reverse Warburg effect, cancer glucose metabolism, lactate shuttle, oxidative stress Received: March 03, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Accepted: April 25, 2017&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Published: May 25, 2017 ABSTRACT Although survival outcomes of cancer patients have been improved dramatically via conventional chemotherapy and targeted therapy over the last decades, there are still some tough clinical challenges that badly needs to be overcome, such as anticancer drug resistance, inevitable recurrences, cancer progression and metastasis. Simultaneously, accumulated evidence demonstrates that aberrant glucose metabolism termed &lsquo;the Warburg effect&rsquo; in cancer cell is closely associated with malignant phenotypes. In 2009, a novel &lsquo;two-compartment metabolic coupling&rsquo; model, also named &lsquo;the reverse Warburg effect&rsquo;, was proposed and attracted lots of attention. Based on this new model, we consider whether this new viewpoint can be exploited for improving the existent anti-cancer therapeutic strategies. Our review focuses on the paradigm shift from &lsquo;the Warburg effect&rsquo; to &lsquo;the reverse Warburg effect&rsquo;, the features and molecular mechanisms of &lsquo;the reverse Warburg effect&rsquo;, and then we discuss its significance in fundamental researches and clinical practice.

BACKGROUND: MicroRNAs (miRNAs) are small non-coding RNAs (~22 nucleotides) that regulate gene expression at a post-transcriptional level via imperfect base pairing to the 3'-UTR of their target mRNAs. Previous studies from our group identified a number of deregulated miRNAs due to the loss of p53 tumor suppressor in colon cancer cell lines. To further investigate the in vivo biological significance of these miRNAs, the expressions of hsa-let-7g, hsa-miR-143, hsa-miR-145, hsa-miR-181b and hsa-miR-200c were investigated using formalin-fixed paraffin-embedded (FFPE) colon cancer specimens to evaluate the potential relationship with chemosensitivity and tumorigenesis. PATIENTS AND METHODS: Forty-six patients with recurrent or residual colon cancer lesions were treated with the 5-fluorouracil-based antimetabolite S-1. This includes twenty-one pairs of tumor and normal samples. Total RNAs were isolated and the expression level of each particular miRNA was quantified using real time qRT-PCR analysis. RESULTS: The expression levels of hsa-let-7g, hsa-miR-181b and hsa-miR-200c were over-expressed in tumor tissues compared to normal tissues. The expression levels of hsa-let-7g (p=0.03; Mann-Whitney test) and hsa-miR-181b (p=0.02; Mann-Whitney test) were strongly associated with clinical response to S-1. Although hsa-let-7g and hsa-miR-181b are strongly associated with patient's response to S-1 treatment, they are not significant prognostic factors for predicting survival. CONCLUSION: hsa-let-7g, hsa-miR-181b and hsa-miR-200c may be associated with tumorigenesis in colon cancer. In addition, hsa-let-7g and hsa-miR-181b may be potential indicators for chemoresponse to S-1 based chemotherapy.

The functions of non-coding microRNAs (miRNAs) in tumorigenesis are just beginning to emerge. Previous studies from our laboratory have identified a number of miRNAs that were deregulated in colon cancer cell lines due to the deletion of the p53 tumor suppressor gene. In this study, the in vivo significance of some of these miRNAs was further evaluated using colorectal clinical samples. Ten miRNAs (hsa-let-7b, hsa-let-7g, hsa-miR-15b, hsa-miR-181b, hsa-miR-191, hsa-miR-200c, hsa-miR-26a, hsa-miR-27a, hsa-miR-30a-5p and hsa-miR-30c) were evaluated for their potential prognostic value in colorectal cancer patients. Forty eight snap frozen clinical colorectal samples (24 colorectal cancer and 24 paired normal patient samples) with detailed clinical follow-up information were selected. The expression levels of 10 miRNAs were quantified via qRT-PCR analysis. The statistical significance of these markers for disease prognosis was evaluated using a two tailed paired Wilcoxon test. A Kaplan-Meier survival curve was generated followed by performing a Logrank test. Among the ten miRNAs, hsa-miR-15b (p = 0.0278), hsa-miR-181b (p = 0.0002), hsa-miR-191 (p = 0.0264) and hsa-miR-200c (p = 0.0017) were significantly over-expressed in tumors compared to normal colorectal samples. Kaplan-Meier survival analysis indicated that hsa-miR-200c was significantly associated with patient survival (p = 0.0122). The patients (n = 15) with higher hsa-miR-200c expression had a shorter survival time (median survival = 26 months) compared to patients (n = 9) with lower expression (median survival = 38 months). Sequencing analysis revealed that hsa-miR-181b (p = 0.0098) and hsa-miR-200c (p = 0.0322) expression were strongly associated with the mutation status of the p53 tumor suppressor gene. Some of these miRNAs may function as oncogenes due to their over-expression in tumors. hsa-miR-200c may be a potential novel prognostic factor in colorectal cancer.

MicroRNAs are small and non-coding RNA molecules with the master role in regulation of gene expression at post-transcriptional/translational levels. Many methods have been developed for microRNA loss-of-function study, such as antisense inhibitors and sponges; however, the robustness, specificity, and stability of these traditional strategies are not highly satisfied. CRISPR/cas9 system is emerging as a novel genome editing tool in biology/medicine research, but its indication in microRNA research has not been studied exclusively. In this study, we clone CRISPR/cas9 constructs with single-guide RNAs specifically targeting biogenesis processing sites of selected microRNAs; and we find that CRISPR/cas9 can robustly and specifically reduce the expression of these microRNAs up to 96%. CRISPR/cas9 also shows an exclusive benefit in control of crossing off-target effect on microRNAs in the same family or with highly conserved sequences. More significantly, for the first time, we demonstrate the long term stability of microRNA knockdown phenotype by CRISPR/cas9 in both in vitro and in vivo models.

CD133 (Prominin-1) is considered the most important cancer stem cell (CSC)-associated marker identified so far, with increased expression in the CSC fraction of a large variety of human malignancies, including melanoma. Here we investigated the effects of CD133 downregulation in vitro and in vivo in human metastatic melanoma. The average number of CD133 molecules on the cell surface of FEMX-I melanoma cells was decreased by 8.7-fold and 1.8-fold using two different short hairpin RNAs. Downregulation of CD133, confirmed by immunocytochemistry, Western blotting, microarray analysis, and reverse transcription-polymerase chain reaction, resulted in slower cell growth, reduced cell motility, and decreased capacity to form spheroids under stem cell-like growth conditions. Clonal analysis revealed that the reduction in growth rate was proportional to the extent of CD133 downregulation. Monoclonal antibodies directed against two different epitopes of the CD133 protein induced a specific, dose-dependent cytotoxic effect in FEMX-I cells. The downregulation of CD133 severely reduced the capacity of the cells to metastasize, particularly to the spinal cord. In the CD133 downregulated cells, microarray analysis revealed expression changes for only 143 annotated genes (76 up- and 67 downregulated). Ten of the 76 upregulated genes coded for Wnt inhibitors, suggesting an interaction between CD133 and the canonical Wnt pathway. We conclude that CD133, in addition to its role as a CSC marker, is an important therapeutic target for metastatic melanoma and, potentially, for other CD133-expressing cancer types.

Honokiol (3',5-di-(2-propenyl)-1,1'-biphenyl-2,4'-diol) is a bioactive natural product derived from Magnolia spp. Recent studies have demonstrated anti-inflammatory, anti-angiogenic, anti-oxidative and anticancer properties of honokiol in vitro and in preclinical models. Honokiol targets multiple signaling pathways including nuclear factor kappa B (NF-κB), signal transducers and activator of transcription 3 (STAT3), epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (m-TOR), which have great relevance during cancer initiation and progression. Furthermore, pharmacokinetic profile of honokiol has revealed a desirable spectrum of bioavailability after intravenous administration in animal models, thus making it a suitable agent for clinical trials. In this review, we discuss recent data describing the molecular targets of honokiol and its anti-cancer activities against various malignancies in pre-clinical models. Evaluation of honokiol in clinical trials will be the next step towards its possible human applications.

To model the heterogeneity of breast cancer as observed in the clinic, we employed an ex vivo model of breast tumor tissue. This methodology maintained the histological integrity of the tumor tissue in unselected breast cancers, and importantly, the explants retained key molecular markers that are currently used to guide breast cancer treatment (e.g., ER and Her2 status). The primary tumors displayed the expected wide range of positivity for the proliferation marker Ki67, and a strong positive correlation between the Ki67 indices of the primary and corresponding explanted tumor tissues was observed. Collectively, these findings indicate that multiple facets of tumor pathophysiology are recapitulated in this ex vivo model. To interrogate the potential of this preclinical model to inform determinants of therapeutic response, we investigated the cytostatic response to the CDK4/6 inhibitor, PD-0332991. This inhibitor was highly effective at suppressing proliferation in approximately 85% of cases, irrespective of ER or HER2 status. However, 15% of cases were completely resistant to PD-0332991. Marker analyses in both the primary tumor tissue and the corresponding explant revealed that cases resistant to CDK4/6 inhibition lacked the RB-tumor suppressor. These studies provide important insights into the spectrum of breast tumors that could be treated with CDK4/6 inhibitors, and defines functional determinants of response analogous to those identified through neoadjuvant studies.