Suman Kanji

Circulating microRNAs in cancer: Hope or hype?

Circulating miRNAs are a novel class of stable, minimally invasive disease biomarkers that are considered to be valuable in diagnosis, prognosis and treatment response monitoring. Unlike intracellular miRNAs, circulating miRNAs are released from their producer cells and, based on their targeted functions, they may shuttle in and out of circulation. Their discovery has opened up new avenues for clinical realms and led to a quest for targeted biomarkers. Subsequently, as more cell-free miRNAs are being discovered, their expression is expected to provide precise information regarding disease progression and treatment outcomes, thereby fostering personalized therapeutic strategies. The significance of circulating miRNAs capitalizes on the fact that they are highly stable in body fluids and their expression levels can be detected by common techniques such as qPCR and microarray. However, discrepancies have started to emerge in terms of their reliability and their response under physiological and pathological conditions. Functional studies are still pending, which may determine whether circulating miRNAs play a role as a central component or just as an auxiliary tuner. Also, the distinct clinical signatures that they display have never been subjected to an extensive critical review and experimental validation. As a consequence, the applicability of circulating miRNAs remains a matter of deliberation, despite many intriguing perspectives about their competency. In this review, we highlight some ambiguous issues with the application of circulating miRNAs, which may warrant an immediate consideration. We propose that the circulating miRNA domain needs to be reevaluated to authenticate their specific role and to probe whether they actually carry any clinical weightage.

Human Umbilical Cord Blood-Derived CD34+ Cells Reverse Osteoporosis in NOD/SCID Mice by Altering Osteoblastic and Osteoclastic Activities

Background Osteoporosis is a bone disorder associated with loss of bone mineral density and micro architecture. A balance of osteoblasts and osteoclasts activities maintains bone homeostasis. Increased bone loss due to increased osteoclast and decreased osteoblast activities is considered as an underlying cause of osteoporosis. Methods and Findings The cures for osteoporosis are limited, consequently the potential of CD34+ cell therapies is currently being considered. We developed a nanofiber-based expansion technology to obtain adequate numbers of CD34+ cells isolated from human umbilical cord blood, for therapeutic applications. Herein, we show that CD34+ cells could be differentiated into osteoblastic lineage, in vitro. Systemically delivered CD34+ cells home to the bone marrow and significantly improve bone deposition, bone mineral density and bone micro-architecture in osteoporotic mice. The elevated levels of osteocalcin, IL-10, GM-CSF, and decreased levels of MCP-1 in serum parallel the improvements in bone micro-architecture. Furthermore, CD34+ cells improved osteoblast activity and concurrently impaired osteoclast differentiation, maturation and functionality. Conclusions These findings demonstrate a novel approach utilizing nanofiber-expanded CD34+ cells as a therapeutic application for the treatment of osteoporosis.

Impact of Diffusion Barriers to Small Cytotoxic Molecules on the Efficacy of Immunotherapy in Breast Cancer

Molecular-focused cancer therapies, e.g., molecularly targeted therapy and immunotherapy, so far demonstrate only limited efficacy in cancer patients. We hypothesize that underestimating the role of biophysical factors that impact the delivery of drugs or cytotoxic cells to the target sites (for associated preferential cytotoxicity or cell signaling modulation) may be responsible for the poor clinical outcome. Therefore, instead of focusing exclusively on the investigation of molecular mechanisms in cancer cells, convection-diffusion of cytotoxic molecules and migration of cancer-killing cells within tumor tissue should be taken into account to improve therapeutic effectiveness. To test this hypothesis, we have developed a mathematical model of the interstitial diffusion and uptake of small cytotoxic molecules secreted by T-cells, which is capable of predicting breast cancer growth inhibition as measured both in vitro and in vivo. Our analysis shows that diffusion barriers of cytotoxic molecules conspire with γδ T-cell scarcity in tissue to limit the inhibitory effects of γδ T-cells on cancer cells. This may increase the necessary ratios of γδ T-cells to cancer cells within tissue to unrealistic values for having an intended therapeutic effect, and decrease the effectiveness of the immunotherapeutic treatment.

Human Ovarian Tumor Cells Escape γδ T Cell Recognition Partly by Down Regulating Surface Expression of MICA and Limiting Cell Cycle Related Molecules

Background Mechanisms of human Vγ2Vδ2 T cell-mediated tumor immunity have yet to be fully elucidated. Methods and Findings At least some tumor cell recognition is mediated by NKG2D-MICA interactions. Herein, by using MTT assay and PI-BrdU co-staining and Western-blot, we show that these Vγ2Vδ2 T cells can limit the proliferation of ovarian tumor cells by down regulation of apoptosis and cell cycle related molecules in tumor cells. Cell-to-cell contact is critical. γδ T cell-resistant, but not susceptible ovarian tumor cells escape γδ T cell-mediated immune recognition by up-regulating pErk1/2, thereby decreasing surface MICA levels. Erk1/2 inhibitor pretreatment or incubation prevents this MICA decrease, while up-regulating key cell cycle related molecules such as CDK2, CDK4 and Cyclin D1, as well as apoptosis related molecules making resistant tumor cells now vulnerable to γδ T cell-mediated lysis. Conclusion These findings demonstrate novel effects of γδT cells on ovarian tumor cells.

Methionine and Kynurenine Activate Oncogenic Kinases in Glioblastoma, and Methionine Deprivation Compromises Proliferation.

Purpose: We employed a metabolomics-based approach with the goal to better understand the molecular signatures of glioblastoma cells and tissues, with an aim toward identifying potential targetable biomarkers for developing more effective and novel therapies. Experimental Design: We used liquid chromatography coupled with mass spectrometry (LC-MS/Q-TOF and LC-MS/QQQ) for the discovery and validation of metabolites from primary and established glioblastoma cells, glioblastoma tissues, and normal human astrocytes. Results: We identified tryptophan, methionine, kynurenine, and 5-methylthioadenosine as differentially regulated metabolites (DRM) in glioblastoma cells compared with normal human astrocytes (NHAs). Unlike NHAs, glioblastoma cells depend on dietary methionine for proliferation, colony formation, survival, and to maintain a deregulated methylome (SAM:SAH ratio). In methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells, expression of MTAP transgene did not alter methionine dependency, but compromised tumor growth in vivo. We discovered that a lack of the kynurenine-metabolizing enzymes kynurenine monooxygenase and/or kynureninase promotes the accumulation of kynurenine, which triggers immune evasion in glioblastoma cells. In silico analysis of the identified DRMs mapped the activation of key oncogenic kinases that promotes tumorigenesis in glioblastoma. We validated this result by demonstrating that the exogenous addition of DRMs to glioblastoma cells in vitro results in oncogene activation as well as the simultaneous downregulation of Ser/Thr phosphatase PP2A. Conclusions: We have connected a four-metabolite signature, implicated in the methionine and kynurenine pathways, to the promotion and maintenance of glioblastoma. Together, our data suggest that these metabolites and their respective metabolic pathways serve as potential therapeutic targets for glioblastoma. Clin Cancer Res; 22(14); 3513–23. ©2016 AACR.

Human Vγ2Vδ2 T cells limit breast cancer growth by modulating cell survival-, apoptosis-related molecules and microenvironment in tumors.

Innate immune system has been known to play an important role in inhibiting the malignant transformation, tumor progression and invasion. However, the mechanistic basis remains ambiguous. Despite polyclonality of human γδ T cells, Vγ2Vδ2 T cell subset was shown to recognize and limit the growth of various tumors at various degrees. The differential recognition of the tumor cells by Vγ2Vδ2 T cells are yet to be defined. Our study reveals that γδ T cells limit in vitro growth of most breast tumor cells, such as SkBr7 (HER2+), MCF7 (ER+) and MDA‐MB‐231 (ER−) by inhibiting their survival and inducing apoptosis, except BrCa‐MZ01 (PR+) cells. To investigate detail mechanisms of antineoplastic effects, we found that cell death was associated with the surface expression levels of MICA/B and ICAM1. Molecular signaling analysis demonstrated that inhibition of cell growth by γδ T cells was associated with the lower expression levels of cell survival‐related molecules such as AKT, ERK and concomitant upregulation of apoptosis‐related molecules, such as PARP, cleaved caspase 3 and tumor suppressor genes PTEN and P53. However, opposite molecular signaling was observed in the resistant cell line after coculture with γδ T cells. In vivo, antineoplastic effects of γδ T cells were also documented, where tumor growth was inhibited due to the downregulation of survival signals, strong induction of apoptotic molecules, disruption of microvasculature and increased infiltration of tumor associated macrophages. These findings reveal that a complex molecular signaling is involved in γδ T cell‐mediated antineoplastic effects.

Nanofiber-expanded human umbilical cord blood–derived CD34+ cell therapy accelerates cutaneous wound closure in NOD/SCID mice

Nanofiber‐expanded human umbilical cord blood–derived CD34+ cell therapy has been shown to have potential applications for peripheral and myocardial ischaemic diseases. However, the efficacies of expanded CD34+ cell therapy for treating cutaneous wounds and its mechanisms of action have yet to be established. Using an excisional wound model in non‐obese diabetic/severe combined immune deficient mice, we show herein that CD34+ cells accelerate the wound‐healing process by enhancing collagen synthesis, and increasing fibroblast cell migration within the wound bed. Concomitantly, reduced levels of matrix metalloproteinase (MMPs) such as MMP1, MMP3, MMP9 and MMP13 were detected in the wound beds of animals treated with CD34+ cells compared with vehicle‐treated controls. CD34+ cells were found to mediate enhanced migration and proliferation of dermal fibroblast cells in vitro. Moreover, CD34+ cells secrete collagen in a serum‐deprived environment. In mechanistic studies, co‐culture of CD34+ cells with primary skin fibroblasts increased the expression of collagen1A1, a component of type 1 collagen, and decreased the expression of MMP1 in fibroblast cells in the presence of a proteasome inhibitor. Finally, CD34+ cell–mediated functions were transcriptionally regulated by the c‐Jun N‐terminal kinases pathway. Collectively, these data provide evidence of therapeutic efficacy and a novel mechanism of nanofiber‐expanded CD34+ cell–mediated accelerated wound healing.

Kruppel-like factor 2 (KLF2) regulates monocyte differentiation and functions in mBSA and IL-1β-induced arthritis.

Kruppel-like factor 2 (KLF2) plays an important role in the regulation of a variety of immune cells, including monocytes. We have previously shown that KLF2 inhibits proinflammatory activation of monocytes. However, the role of KLF2 in arthritis is yet to be investigated. In the current study, we show that recruitment of significantly greater numbers of inflammatory subset of CD11b(+)F4/80(+)Ly6C+ monocytes to the inflammatory sites in KLF2 hemizygous mice compared to the wild type littermate controls. In parallel, inflammatory mediators, MCP-1, Cox-2 and PAI-1 were significantly up-regulated in bone marrow-derived monocytes isolated from KLF2 hemizygous mice, in comparison to wild-type controls. Methylated-BSA and IL-1β-induced arthritis was more severe in KLF2 hemizygous mice as compared to the littermate wild type controls. Consistent with this observation, monocytes isolated from KLF2 hemizygous mice showed an increased number of cells matured and differentiated towards osteoclastic lineage, potentially contributing to the severity of cartilage and bone damage in induced arthritic mice. The severity of arthritis was associated with the higher expression of proteins such as HSP60, HSP90 and MMP13 and attenuated levels of pPTEN, p21, p38 and HSP25/27 molecules in bone marrow cells of arthritic KLF2 hemizygous mice compared to littermate wild type controls. The data provide new insights and evidences of KLF2-mediated transcriptional regulation of arthritis via modulation of monocyte differentiation and function.

Plasticity and maintenance of hematopoietic stem cells during development.

Maintenance of hematopoietic stem cells (HSCs) pool depends on fine balance between self-renewal and differentiation of HSCs. HSCs normally reside within the bone marrow niche of an adult mammal. The embryonic development of HSCs is a complex process that involves the migration of developing HSCs in multiple anatomical sites. Throughout the process, developing HSCs receive internal (transcriptional program) and external (HSC niche) signals, which direct them to maintain balance between self-renewal and differentiation, also to generate a pool of HSCs. In physiological condition HSCs differentiate into all mature cell types present in the blood. However, in pathological condition they may differentiate into non-hematological cells according to the need of the body. It was shown that HSCs can transdifferentiate into cell types that do not belong to the hematopoietic system suggests a complete paradigm shift of the hierarchical hematopoietic tree. This review describes the developmental origins and regulation of HSCs focusing on developmental signals that induce the adult hematopoietic stem cell program, as these informations are very critical for manipulating conditions for expansion of HSCs in ex vivo condition. This review also states clinical application and related patents using HSC.

Advances of Stem Cell Therapeutics in Cutaneous Wound Healing and Regeneration

Cutaneous wound healing is a complex multiple phase process, which overlaps each other, where several growth factors, cytokines, chemokines, and various cells interact in a well-orchestrated manner. However, an imbalance in any of these phases and factors may lead to disruption in harmony of normal wound healing process, resulting in transformation towards chronic nonhealing wounds and abnormal scar formation. Although various therapeutic interventions are available to treat chronic wounds, current wound-care has met with limited success. Progenitor stem cells possess potential therapeutic ability to overcome limitations of the present treatments as it offers accelerated wound repair with tissue regeneration. A substantial number of stem cell therapies for cutaneous wounds are currently under development as a result of encouraging preliminary findings in both preclinical and clinical studies. However, the mechanisms by which these stem cells contribute to the healing process have yet to be elucidated. In this review, we emphasize on the major treatment modalities currently available for the treatment of the wound, role of various interstitial stem cells and exogenous adult stem cells in cutaneous wound healing, and possible mechanisms involved in the healing process.