Glycosylation in bladder cancer

Complex carbohydrates are major components of the cell membrane and they play crucial roles in cell-cell and cell-extracellular matrix interactions, as well as in signal transduction. They consist of three kinds of molecular species; glycoproteins, proteoglycans, and glycosphingolipids. There is a distinct difference in carbohydrate profiles between normal and tumor tissues. The characteristic carbohydrate expression associated with malignant transformation is caused by "aberrant glycosylation" catalyzed by specific glycosyltransferases and glycosidases. A close relationship between blood type antigens and bladder cancer was first established in the 1960s, using the classic red-cell adherence test. Lectin immunohistochemical staining eventually replaced the red-cell adherence test. In the 1980s, several monoclonal antibodies were raised against complex carbohydrates, and the clinico-pathologic significance of blood type antigens in bladder cancer was investigated using these antibodies. Recent studies have demonstrated the high sensitivity and specificity of immunostaining for Lewis X antigen, a carbohydrate blood type antigen, in exfoliated cells from voided urine samples. Other than blood type antigens, the significance of aberrant glycosylation in bladder cancer has been demonstrated in a number of articles. For instance, overexpression of the ganglioside (an acidic glycosphingolipid which has sialic acid) GM3 induces apoptosis and reduces invasive potential in a bladder cancer cell line. Hyaluronic acid promotes tumor metastasis and is an accurate diagnostic marker for bladder cancer. The expression of N-acetylglucosaminyltransferase V and beta,1-6 branching N-linked oligosaccharides is closely related to low malignant potential in bladder cancer. Selectins and galectins, specific ligands for carbohydrate antigens, are also key molecules involved in the apoptosis and metastasis of cancer cells. Thus, proteoglycans, glycoproteins, and glycosphingolipids, and their ligands, play crucial roles in the malignant transformation, invasion, and metastasis of bladder cancer. A novel diagnostic and therapeutic approach may be possible by taking advantage of innovative techniques in glycobiology.     

Immune checkpoint blockade opens an avenue of cancer immunotherapy with a potent clinical efficacy

Recent progress in tumor immunology has revealed that tumors generate immunologically restrained milieu during the process of their growth, which facilitates the escape of tumors from host immune systems. Immune checkpoint molecules, which transduce co‐inhibitory signals to immuno‐competent cells, are one of the most important components conferring the immunosuppressive capacity in the tumor microenvironment. Cytotoxic T‐lymphocyte‐associated protein 4 (CTLA‐4) and programmed cell death‐1 (PD‐1) are typical immune checkpoint molecules intimately involved in the suppression of anti‐tumor immunity. Antibodies against those molecules have been developed, such as ipilimumab (anti‐CTLA‐4 antibody), nivolumab and pembrolizumab (anti‐PD‐1 antibody), and have been approved by regulatory agencies and used in some countries. Treatment with these antibodies demonstrates previously unobserved clinical efficacies superior to the conventional therapies. In this review, we first discuss the escape mechanisms of cancer from host immune systems, and then focus on the recent advances in immune checkpoint blockade therapy and on the new findings of related immune reactions, aiming to provide a better understanding of the novel cancer immunotherapies.     

Proximity proteomics identifies cancer cell membrane cis‐molecular complex as a potential cancer target

Cancer‐specific antigens expressed in the cell membrane have been used as targets for several molecular targeted strategies in the last 20 years with remarkable success. To develop more effective cancer treatments, novel targets and strategies for targeted therapies are needed. Here, we examined the cancer cell membrane‐resident “cis‐bimolecular complex” as a possible cancer target (cis‐bimolecular cancer target: BiCAT) using proximity proteomics, a technique that has attracted attention in the last 10 years. BiCAT were detected using a previously developed method termed the enzyme‐mediated activation of radical source (EMARS), to label the components proximal to a given cell membrane molecule. EMARS analysis identified some BiCAT, such as close homolog of L1 (CHL1), fibroblast growth factor 3 (FGFR3) and α2 integrin, which are commonly expressed in mouse primary lung cancer cells and human lung squamous cell carcinoma cells. Analysis of cancer specimens from 55 lung cancer patients revealed that CHL1 and α2 integrin were highly co–expressed in almost all cancer tissues compared with normal lung tissues. As an example of BiCAT application, in vitro simulation of effective drug combinations used for multiple drug treatment strategies was performed using reagents targeted to BiCAT molecules. The combination treatment based on BiCAT information moderately suppressed cancer cell proliferation compared with single administration, suggesting that the information about BiCAT in cancer cells is useful for the appropriate selection of the combination among molecular targeted reagents. Thus, BiCAT has the potential to contribute to several molecular targeted strategies in future.     

Immuno‐based detection of extracellular vesicles in urine as diagnostic marker for prostate cancer

Extracellular vesicles (including the subclass exosomes) secreted by cells contain specific proteins and RNA that could be of interest in determining new markers. Isolation/characterization of PCa‐derived exosomes from bodily fluids enables us to discover new markers for this disease. Unfortunately, isolation with current techniques (ultracentrifugation) is labor intensive and other techniques are still under development. The goal of our study was to develop a highly sensitive time‐resolved fluorescence immunoassay (TR‐FIA) for capture/detection of PCa‐derived exosomes. In our assay, biotinylated capture antibodies against human CD9 or CD63 were incubated on streptavidin‐coated wells. After application of exosomes, Europium‐labeled detection antibodies (CD9 or CD63) were added. Cell medium from 37 cell lines was taken to validate this TR‐FIA. Urine was collected (after digital rectal exam) from patients with PCa (n = 67), men without PCa (n = 76). As a control, urine was collected from men after radical prostatectomy (n = 13), women (n = 16) and patients with prostate cancer without digital rectal exam (n = 16). Signal intensities were corrected for urinary PSA and creatinine. This TR‐FIA can measure purified exosomes with high sensitivity and minimal background signals. Exosomes can be measured in medium from 37 cell lines and in urine. DRE resulted in a pronounced increase in CD63 signals. After DRE and correction for urinary PSA, CD9 and CD63 were significantly higher in men with PCa. This TR‐FIA enabled us to measure exosomes with high sensitivity directly from urine and cell medium. This TR‐FIA forms the basis for testing different antibodies directed against exosome membrane markers to generate disease‐specific detection assays.     

胃癌淋巴结转移相关分子标志物的研究进展

复发和转移是导致胃癌死亡率高居不下的主要原因,淋巴结转移是胃癌转移的一个早期事件和独立的预后因素。胃癌淋巴结转移过程可能涉及到许多分子作用机制和信号传导途径,包括肿瘤淋巴管生成相关基因、细胞粘附分子、细胞外基质相关蛋白、细胞因子、肿瘤转移相关基因等。本文综述胃癌淋巴结转移的相关分子标志物的研究进展。     

与食管癌相关的癌症干细胞膜标志物的研究进展

食管癌发病率和死亡率在癌症中均位于前列。食管癌预后较差,尽管目前治疗手段不断进步,也是难治疗和治愈的恶性肿瘤之一。当前,针对食管肿瘤遗传学改变方面的研究正在逐步进行,包括分子机制、基因表达及肿瘤干细胞等的研究。本文就与食管癌相关的癌症干细胞膜标志物的研究进展作一综述。     

Membrane proteases as potential diagnostic and therapeutic targets for breast malignancy

Summary Metastasizing cancer cells can invade the extracellular matrix using plasma membrane protrusions, termed invadopodia, that contact and dissolve the matrix. Various membrane associated proteases localized on the invadopodial membranes are responsible for the extracellular matrix degradation. Work from our laboratory shows that secreted proteases including Gelatinase A, and high molecular weight integral membrane proteases are associated with cell surface invadopodia. Three cell types, including chicken embryonic cells transformed by Rous sarcoma virus, human malignant melanoma cell line LOX, and human breast carcinoma cell line MDA-MB-231, retain the invasive phenotypein vitro, express invadopodia, degrade and enter into a fibronectin-rich collagenous matrix. We suggest that invadopodium-associated proteases are ideal targets for the diagnosis and treatment of cancer as their presence in association with primary tumors may signal increased metastatic potential. An approach toward the development of new prognostic markers for breast malignancy involved production of monoclonal antibodies directed against membrane proteases in a mixture of glycoproteins. Double immunofluorescent technique using a known invadopodium marker is designed to select specific monoclonal antibodies colocalizing at the invasion front, on invadopodia of cancer cells. Membrane protease accessibility at the cell surface can therefore be exploited for therapeutic advances by the development of specific antibodies and inhibitors that block their activities, and by the use of monoclonal antibodies to target cytotoxic molecules to micrometastases. Also, this same accessibility may potentially be used to detect surface proteases on micrometastases or to detect components shed by micrometastases in serum.     

Therapy‐induced enrichment of putative lung cancer stem‐like cells

Tumour drug resistance is a major issue in the management of lung cancer patients as almost all lung tumours are either intrinsically resistant or quickly develop acquired resistance to chemotherapeutic drugs. Cancer drug resistance has recently been linked, at least in part, to the existence of cancer stem‐like cells (CSLCs), a small sub‐population of cells within the tumour that possess stem‐like properties. CSLCs are often isolated by fluorescence activated cell sorting (FACS) according to the expression of certain stem‐like cell membrane markers. Conflicting results regarding the specificity of particular stem cell surface markers for isolating CSLCs have, however, been recently reported. Therefore, alternative strategies enabling the identification and study of CSLCs should be considered, particularly in tumour types where appropriate stem cell markers are not well established and validated, like in lung cancer. In this article, we review data indicating therapy‐selection as a valid approach for putative lung CSLCs enrichment. We believe that this strategy would be determinant for correctly assessing and characterising the sub‐populations of CSLCs that are able to survive chemo or radiotherapy regimens and, at the same time, also have the ability to recapitulate and sustain tumour growth. Using therapy‐induced enrichment of CSLCs may, therefore, prove to be an extremely useful method for studying CSLCs and provide new clues regarding potential therapeutic targets for their efficient elimination, which will undoubtedly play a decisive role in improving lung cancer patients' survival.     

Vaccination with human amniotic epithelial cells confer effective protection in a murine model of Colon adenocarcinoma

As a prophylactic cancer vaccine, human amniotic membrane epithelial cells (hAECs) conferred effective protection in a murine model of colon cancer. The immunized mice mounted strong cross‐protective CTL and antibody responses. Tumor burden was significantly reduced in tumor‐bearing mice after immunization with hAECs. Placental cancer immunotherapy could be a promising approach for primary prevention of cancer. In spite of being the star of therapeutic strategies for cancer treatment, the results of immunotherapeutic approaches are still far from expectations. In this regard, primary prevention of cancer using prophylactic cancer vaccines has gained considerable attention. The immunologic similarities between cancer development and placentation have helped researchers to unravel molecular mechanisms responsible for carcinogenesis and to take advantage of stem cells from reproductive organs to elicit robust anti‐cancer immune responses. Here, we showed that vaccination of mice with human amniotic membrane epithelial cells (hAECs) conferred effective protection against colon cancer and led to expansion of systemic and splenic cytotoxic T cell population and induction of cross‐protective cytotoxic responses against tumor cells. Vaccinated mice mounted tumor‐specific Th1 responses and produced cross‐reactive antibodies against cell surface markers of cancer cells. Tumor burden was also significantly reduced in tumor‐bearing mice immunized with hAECs. Our findings pave the way for potential future application of hAECs as an effective prophylactic cancer vaccine.     

Secondary malignancies in pediatric cancer survivors: Perspectives and review of the literature

With continuing improvements in the successful treatment of pediatric malignancies, long term survivors of pediatric cancers and their providers are faced with new oncologic issues regarding long‐term morbidities. As pediatric cancer survivors have matured into adulthood, the development of secondary malignancies has become a significant issue for these patients. Whether a consequence of treatment for the patient's original cancer, such as chemotherapy, ionizing radiation, or hematopoietic stem cell transplantation, secondary malignancies now present patients and providers with new challenges regarding treatment, surveillance and counseling. We review the major risk factors for secondary malignancies in pediatric cancer survivors, with particular emphasis on important molecular and cytogenetic risk factors, both inherited and acquired. We conclude with a discussion of recommendations for surveillance and counseling of these patients.     

Targeting cancer stem‐like cells in glioblastoma and colorectal cancer through metabolic pathways

Cancer stem‐like cells (CSCs) are thought to be the main cause of tumor occurrence, progression and therapeutic resistance. Strong research efforts in the last decade have led to the development of several tailored approaches to target CSCs with some very promising clinical trials underway; however, until now no anti‐CSC therapy has been approved for clinical use. Given the recent improvement in our understanding of how onco‐proteins can manipulate cellular metabolic networks to promote tumorigenesis, cancer metabolism research may well lead to innovative strategies to identify novel regulators and downstream mediators of CSC maintenance. Interfering with distinct stages of CSC‐associated metabolics may elucidate novel, more efficient strategies to target this highly malignant cell population. Here recent discoveries regarding the metabolic properties attributed to CSCs in glioblastoma (GBM) and malignant colorectal cancer (CRC) were summarized. The association between stem cell markers, the response to hypoxia and other environmental stresses including therapeutic insults as well as developmentally conserved signaling pathways with alterations in cellular bioenergetic networks were also discussed. The recent developments in metabolic imaging to identify CSCs were also summarized. This summary should comprehensively update basic and clinical scientists on the metabolic traits of CSCs in GBM and malignant CRC.     

Cell‐based selection provides novel molecular probes for cancer stem cells

Cancer stem cells (CSC) represent a malignant subpopulation of cells in hierarchically organized tumors. They constitute a subpopulation of malignant cells within a tumor mass and possess the ability to self‐renew giving rise to heterogeneous tumor cell populations with a complex set of differentiated tumor cells. CSC may be the cause of metastasis and therapeutic refractory disease. Because few markers exist to identify and isolate pure CSC, we used cell‐based Systematic Evolution of Ligands by EXponential enrichment (cell‐SELEX) to create DNA aptamers that can identify novel molecular targets on the surfaces of live CSC. Out of 22 putative DNA sequences, 3 bound to ～90% and 5 bound to ～15% of DU145 prostate cancer cells. The 15% of cells that were positive for the second panel of aptamers expressed high levels of E‐cadherin and CD44, had high aldehyde dehydrogenase 1 activity, grew as spheroids under nonadherent culture conditions, and initiated tumors in immune‐compromised mice. The discovery of the molecular targets of these aptamers could reveal novel CSC biomarkers.     

Oncogenes and oncoproteins as tumor markers.

A major problem in the management of patients with cancer is the lack of specific tumor markers for the early detection, the accurate prediction of biological behavior and for accurate assessment of prognosis. A new and exciting answer to this problem may now become available following the discovery of specific genes associated with malignancy. The role of such genes and their products are now being identified and their role in cancer is under intense investigation. On a clinical level, these genes and their products may allow us to improve our understanding of disease etiology and provide more precise diagnostic, prognostic and therapeutic characterization of individual tumors. This paper discusses the possibilities of using the altered expression of oncogenes and tumor suppressor genes and their products in neoplastic tissues as markers for the diagnosis and prognosis of malignant disease. These data support the view that detailed analysis of such gene expression has the potential to predict a tumor's behavior as well as the response to different treatment modalities.     

The melanoma specific 9.2.27PE immunotoxin efficiently kills melanoma cells in vitro

Malignant melanomas are generally drug resistant and have a very poor prognosis. We have studied the effects of a chemical conjugate of pseudomonas exotoxin A (PE) and the antibody 9.2.27, which recognizes the high molecular weight melanoma associated antigen (HMW‐MAA) expressed in most malignant melanomas and melanoma cell lines. We demonstrate that the 9.2.27PE immunotoxin (IT) induces cell death in malignant melanoma cells through protein synthesis inhibition followed by some morphological and biochemical features of apoptosis, like rounding up of cells, chromatin condensation and inactivation of PARP. Unlike previous results with the 425.3PE IT in breast cancer cells, we detected no depolarization of the mitochondrial membrane after 9.2.27PE IT treatment. This is likely due to the lack of strong activation of caspase‐8 and caspase‐3. The lack of depolarization suggests that cytochrome c, a molecule that triggers activation of caspase‐3, was retained within the mitochondria. In addition, the protein level of the antiapoptotic Bcl‐2 did not decrease in contrast to other antiapoptotic molecules belonging to the inhibitor of apoptosis and the Bcl‐2 family. This suggests that Bcl‐2 may play a role in maintaining the mitochondrial membrane integrity in the 9.2.27PE‐treated cells. Nevertheless, 9.2.27PE IT efficiently killed malignant melanoma cells that can be ascribed to inhibition of protein synthesis followed by some morphological and biochemical features of apoptosis. © 2009 UICC     

Adhesion molecule protein signature in ovarian cancer effusions is prognostic of patient outcome

BACKGROUND:

Ovarian cancer cells in malignant effusions lack attachment to solid‐phase matrix substrata and receive survival stimuli through cell–cell and cell–soluble matrix molecule interactions. We hypothesized that adhesion‐related survival and proliferation pathway signals can inform clinical outcomes and guide targeted therapeutics.

METHODS:

Lysed cell pellets from a blinded set of benign (n = 20) and malignant (n = 51) peritoneal and pleural ovarian cancer patient effusions were applied to reverse‐phase protein arrays and examined using validated antibodies to adhesion‐associated protein endpoints. Results were subjected to hierarchical clustering for signature development. Association between specimen type, protein expression, and clinicopathologic associations were analyzed using the Mann‐Whitney U test. Survival outcomes were estimated using the Kaplan‐Meier method with log‐rank comparison.

RESULTS:

A cell adhesion protein signature obtained from unsupervised clustering distinguished malignant from benign effusions (P = 6.18E‐06). Protein subset analyses from malignant cases defined 3 cell adhesion protein clusters driven by E‐cadherin, epithelial cell adhesion molecule, and N‐cadherin, respectively. The components of the E‐ and N‐cadherin clusters correlated with clinical outcome by Kaplan‐Meier statistics. Univariate analysis indicated that FAK and phosphorylated AKT were associated with higher overall and progression‐free survival (PFS) (P = .03), and Akt, phosphorylated paxillin, and E‐ and N‐cadherin were associated with improved PFS (P ≤ .05). If 4 or 5 of the index adhesion proteins were high, PFS was improved by multivariate analysis (P ≤ .01).

CONCLUSIONS:

This hypothesis‐testing examination of tumor cell adhesion molecules and pathways yielded potential predictive biomarkers with which to triage patients to selected molecular therapeutics and may serve as a platform for biomarker‐based stratification for clinical application. Cancer 2011;. Published 2011 by the American Cancer Society.