Manish V. Bais

BMP2 Is Essential for Post Natal Osteogenesis but Not for Recruitment of Osteogenic Stem Cells

The effects of BMP2 on bone marrow stromal cell differentiation and bone formation after bone marrow ablation were determined using C57 BL/6J (B6) mice. Inhibition of BMP2 expression with lentiviral BMP2 shRNA prevented both mineralized nodule formation in vitro and bone formation in vivo, and blocked the expression of Runx2 and osterix, transcriptional determinants of terminal osteogenic differentiation. No effect was observed on the expression of Sox9, a transcription factor, which is the one of the first transcriptional determinant to be expressed in committed chondroprogenitor and osteoprogenitor cells. In vitro studies showed that exogenously added BMP7 rescued the expression of osterix and enhanced the expression of Sox9, but had no effect on the expression of Runx2, while it only partially recovered the development of mineral deposition in the cultures. On the other hand, the exogenous addition of BMP2 rescued both Runx2 and osterix expression, did not enhance the expression of Sox9, but fully recovered the inhibition of mineral deposition in the cultures. Using antibodies against CD146 and Sox9, immunohistological examination of the cell populations found in the medullary space three days after bone marrow ablation, showed qualitatively equal numbers of cells expressing these skeletal progenitor and stem cell markers in control and BMP2 shRNA treated animals. Fluorescence Activated Cell Sorting (FACS) analysis of the cells found with the marrow cavities at three days after marrow ablation using CD146 antibody showed near equal numbers of immunopositive cells in both control and shRNA treated animals. In summary, the differences observed in vitro for BMP2 and BMP7 on osteogenic gene expression and mineralization suggest that they have differing effects on bone cell differentiation. These results further demonstrate that in vivo BMP2 is a central morphogenetic regulator of post natal osteoprogenitor differentiation, but does not affect recruitment of progenitors to the osteoblastic lineage.

Transcriptional Analysis of Fracture Healing and the Induction of Embryonic Stem Cell-Related Genes

Fractures are among the most common human traumas. Fracture healing represents a unique temporarily definable post-natal process in which to study the complex interactions of multiple molecular events that regulate endochondral skeletal tissue formation. Because of the regenerative nature of fracture healing, it is hypothesized that large numbers of post-natal stem cells are recruited and contribute to formation of the multiple cell lineages that contribute to this process. Bayesian modeling was used to generate the temporal profiles of the transcriptome during fracture healing. The temporal relationships between ontologies that are associated with various biologic, metabolic, and regulatory pathways were identified and related to developmental processes associated with skeletogenesis, vasculogenesis, and neurogenesis. The complement of all the expressed BMPs, Wnts, FGFs, and their receptors were related to the subsets of transcription factors that were concurrently expressed during fracture healing. We further defined during fracture healing the temporal patterns of expression for 174 of the 193 genes known to be associated with human genetic skeletal disorders. In order to identify the common regulatory features that might be present in stem cells that are recruited during fracture healing to other types of stem cells, we queried the transcriptome of fracture healing against that seen in embryonic stem cells (ESCs) and mesenchymal stem cells (MSCs). Approximately 300 known genes that are preferentially expressed in ESCs and ∼350 of the known genes that are preferentially expressed in MSCs showed induction during fracture healing. Nanog, one of the central epigenetic regulators associated with ESC stem cell maintenance, was shown to be associated in multiple forms or bone repair as well as MSC differentiation. In summary, these data present the first temporal analysis of the transcriptome of an endochondral bone formation process that takes place during fracture healing. They show that neurogenesis as well as vasculogenesis are predominant components of skeletal tissue formation and suggest common pathways are shared between post-natal stem cells and those seen in ESCs.

A YAP/TAZ-Regulated Molecular Signature Is Associated with Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) is a prevalent form of cancer that develops from the epithelium of the oral cavity. OSCC is on the rise worldwide, and death rates associated with the disease are particularly high. Despite progress in understanding the mutational and expression landscape associated with OSCC, advances in deciphering these alterations for the development of therapeutic strategies have been limited. Further insight into the molecular cues that contribute to OSCC is therefore required. Here, we show that the transcriptional regulators YAP (YAP1) and TAZ (WWTR1), which are key effectors of the Hippo pathway, drive protumorigenic signals in OSCC. Regions of premalignant oral tissues exhibit aberrant nuclear YAP accumulation, suggesting that dysregulated YAP activity contributes to the onset of OSCC. Supporting this premise, we determined that nuclear YAP and TAZ activity drives OSCC cell proliferation, survival, and migration in vitro, and is required for OSCC tumor growth and metastasis in vivo. Global gene expression profiles associated with YAP and TAZ knockdown revealed changes in the control of gene expression implicated in protumorigenic signaling, including those required for cell cycle progression and survival. Notably, the transcriptional signature regulated by YAP and TAZ significantly correlates with gene expression changes occurring in human OSCCs identified by The Cancer Genome Atlas (TCGA), emphasizing a central role for YAP and TAZ in OSCC biology. Implications: This study defines a YAP/TAZ-regulated transcriptional program in OSCC and reveals novel roles for nuclear YAP/TAZ activity in the onset and progression of this cancer. Mol Cancer Res; 13(6); 957–68. ©2015 AACR.

Recombinant Lysyl Oxidase Propeptide Protein Inhibits Growth and Promotes Apoptosis of Pre-Existing Murine Breast Cancer Xenografts

Lysyl oxidase propeptide (LOX-PP) ectopic overexpression inhibits the growth of cancer xenografts. Here the ability and mode of action of purified recombinant LOX-PP (rLOX-PP) protein to inhibit the growth of pre-existing xenografts was determined. Experimental approaches employed were direct intratumoral injection (i.t.) of rLOX-PP protein into murine breast cancer NF639 xenografts, and application of a slow release formulation of rLOX-PP implanted adjacent to tumors in NCR nu/nu mice (n = 10). Tumors were monitored for growth, and after sacrifice were subjected to immunohistochemical and Western blot analyses for several markers of proliferation, apoptosis, and for rLOX-PP itself. Direct i.t. injection of rLOX-PP significantly reduced tumor volume on days 20, 22 and 25 and tumor weight at harvest on day 25 by 30% compared to control. Implantation of beads preloaded with 35 micrograms rLOX-PP (n = 10) in vivo reduced tumor volume and weight at sacrifice when compared to empty beads (p<0.05). A 30% reduction of tumor volume on days 22 and 25 (p<0.05) and final tumor weight on day 25 (p<0.05) were observed with a reduced tumor growth rate of 60% after implantation. rLOX-PP significantly reduced the expression of proliferation markers and Erk1/2 MAP kinase activation, while prominent increases in apoptosis markers were observed. rLOX-PP was detected by immunohistochemistry in harvested rLOX-PP tumors, but not in controls. Data provide pre-clinical findings that support proof of principle for the therapeutic anti-cancer potential of rLOX-PP protein formulations.

Time course of Newcastle disease virus-induced apoptotic pathways.

Newcastle disease virus (NDV) causes economically significant Newcastle disease (ND) in almost all birds worldwide. Previous studies have shown that NDV induces caspase dependent apoptotic pathways in infected cells. In the present study, time course induction of apoptotic pathways in Vero cells is described. In NDV-infected cells, caspase-8 activity, percentage of cells showing TRAIL expression was higher at 24h p.i. (post-infection) compared to 48 h p.i. In contrast, caspase-9 activity, efflux of cytochrome c, loss of mitochondrial membrane potential was higher at 48 h compared to 24h p.i. The caspase-3 activity was high both times. Based on these results, it was concluded that at 24h p.i., NDV induces apoptosis through extrinsic apoptotic pathway while at 48 h p.i. predominantly through intrinsic apoptotic pathway.

Lysyl Oxidase-like-2 (LOXL2) Is a Major Isoform in Chondrocytes and Is Critically Required for Differentiation

The lysyl oxidase family is made up of five members: lysyl oxidase (LOX) and lysyl oxidase-like 1–4 (LOXL1-LOXL4). All members share conserved C-terminal catalytic domains that provide for lysyl oxidase or lysyl oxidase-like enzyme activity; and more divergent propeptide regions. LOX family enzyme activities catalyze the final enzymatic conversion required for the formation of normal biosynthetic collagen and elastin cross-links. The importance of lysyl oxidase enzyme activity to normal bone development has long been appreciated, but regulation and roles for specific LOX isoforms in bone formation in vivo is largely unexplored. Fracture healing recapitulates aspects of endochondral bone development. The present study first investigated the expression of all LOX isoforms in fracture healing. A remarkable coincidence of LOXL2 expression with the chondrogenic phase of fracture healing was found, prompting more detailed analyses of LOXL2 expression in normal growth plates, and LOXL2 expression and function in developing ATDC5 chondrogenic cells. Data show that LOXL2 is expressed by pre-hypertrophic and hypertrophic chondrocytes in vivo, and that LOXL2 expression is regulated in vitro as a function of chondrocyte differentiation. Moreover, LOXL2 knockdown studies in vitro show that LOXL2 expression is required for ATDC5 chondrocyte cell line differentiation through regulation of SNAIL and SOX9, important transcription factors that control chondrocyte differentiation. Taken together, data provide evidence that LOXL2, like LOX, is a multifunctional protein. LOXL2 promotes chondrocyte differentiation by mechanisms that are likely to include roles as both a regulator and an effector of chondrocyte differentiation.

Role of Nanog in the Maintenance of Marrow Stromal Stem Cells during Post Natal Bone Regeneration

Post natal bone repair elicits a regenerative mechanism that restores the injured tissue to its pre-injury cellular composition and structure and is believed to recapitulate the embryological processes of bone formation. Prior studies showed that Nanog, a central epigenetic regulator associated with the maintenance of embryonic stem cells (ESC) was transiently expressed during fracture healing, Bais et al. In this study, we show that murine bone marrow stromal cells (MSCs) before they are induced to undergo osteogenic differentiation express ∼50× the background levels of Nanog seen in murine embryonic fibroblasts (MEFs) and the W20-17 murine marrow stromal cell line stably expresses Nanog at ∼80× the MEF levels. Nanog expression in this cell line was inhibited by BMP7 treatment and Nanog lentivrial shRNA knockdown induced the expression of the terminal osteogenic gene osteocalcin. Lentivrial shRNA knockdown or lentiviral overexpression of Nanog in bone MSCs had inverse effects on proliferation, with knockdown decreasing and overexpression increasing MSC cell proliferation. Surgical marrow ablation of mouse tibia by medullary reaming led to a ∼3-fold increase in Nanog that preceded osteogenic differentiation during intramembranous bone formation. Lentiviral shRNA knockdown of Nanog after surgical ablation led to an initial overexpression of osteogenic gene expression with no initial effect on bone formation but during subsequent remodeling of the newly formed bone a ∼50% decrease was seen in the expression of terminal osteogenic gene expression and a ∼50% loss in trabecular bone mass. This loss of bone mass was accompanied by an increased ∼2- to 5-fold adipogenic gene expression and observed increase of fat cells in the marrow space. In summary these data show that Nanog is expressed during surgically induced marrow bone formation and is functionally involved in post natal marrow stromal cell maintenance and differentiation.

Role for the Aryl Hydrocarbon Receptor and Diverse Ligands in Oral Squamous Cell Carcinoma Migration and Tumorigenesis

Over 45,000 new cases of oral and pharyngeal cancers are diagnosed and account for over 8,000 deaths a year in the United States. An environmental chemical receptor, the aryl hydrocarbon receptor (AhR), has previously been implicated in oral squamous cell carcinoma (OSCC) initiation as well as in normal tissue-specific stem cell self-renewal. These previous studies inspired the hypothesis that the AhR plays a role in both the acquisition and progression of OSCC, as well as in the formation and maintenance of cancer stem-like cells. To test this hypothesis, AhR activity in two oral squamous cell lines was modulated with AhR prototypic, environmental and bacterial AhR ligands, AhR-specific inhibitors, and phenotypic, genomic and functional characteristics were evaluated. The data demonstrate that: (i) primary OSCC tissue expresses elevated levels of nuclear AhR as compared with normal tissue, (ii) AhR mRNA expression is upregulated in 320 primary OSCCs, (iii) AhR hyperactivation with several ligands, including environmental and bacterial ligands, significantly increases AhR activity, ALDH1 activity, and accelerates cell migration, (iv) AhR inhibition blocks the rapid migration of OSCC cells and reduces cell chemoresistance, (v) AhR knockdown inhibits tumorsphere formation in low adherence conditions, and (vi) AhR knockdown inhibits tumor growth and increases overall survival in vivo. These data demonstrate that the AhR plays an important role in development and progression of OSCC, and specifically cancer stem-like cells. Prototypic, environmental, and bacterial AhR ligands may exacerbate OSCC by enhancing expression of these properties. Implications: This study, for the first time, demonstrates the ability of diverse AhR ligands to regulate AhR activity in oral squamous cell carcinoma cells, as well as regulate several important characteristics of oral cancer stem cells, in vivo and in vitro. Mol Cancer Res; 14(8); 696–706. ©2016 AACR.

Determination of cell uptake pathways for tumor inhibitor lysyl oxidase propeptide

The lysyl oxidase propeptide (LOX‐PP) is derived from pro‐lysyl oxidase (Pro‐LOX) by extracellular biosynthetic proteolysis. LOX‐PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX‐PP have been identified, LOX‐PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX‐PP (rLOX‐PP) is PI3K‐dependent macropinocytosis in PWR‐1E, PC3, SCC9, MDA‐MB‐231 cell lines. A secondary pathway appears to be dynamin‐ and caveola dependent. The ionic properties of highly basic rLOX‐PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX‐PP, which serves to limit endosomal acidification, sustains PI3K‐dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX‐PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake.

Orthotopic non-metastatic and metastatic oral cancer mouse models

Oral cancer is characterized by high morbidity and mortality with a predisposition to metastasize to different tissues, including lung, liver, and bone. Despite progress in the understanding of mutational profiles and deregulated pathways in oral cancer, patient survival has not significantly improved over the past decades. Therefore, there is a need to establish in vivo models that recapitulate human oral cancer metastasis to evaluate therapeutic potential of novel drugs. Here we report orthotopic tongue cancer nude mouse models to study oral cancer growth and metastasis using human metastatic (UMSCC2) and non-metastatic (CAL27) cell lines, respectively. Transduction of these cell lines with lentivirus expressing red fluorescent protein (DsRed) followed by injection into tongues of immunodeficient mice generated orthotopic tongue tumors that could be monitored for growth and metastasis by fluorescence measurement with an in vivo Imaging System (IVIS 200). The growth rates of CAL27-DsRed induced tumors were higher than UMSCC2-DsRed tumors after day 15, while UMSCC2-DsRed tumors revealed metastasis beginning on day 21. Importantly, UMSCC2 tumors metastasized to a number of tissues including the submandibular gland, lung, kidney, liver, and bone. Further, immunohistochemical analyses of tongue tumors induced by CAL27 and UMSCC2 cells revealed elevated expression of components of protumorigenic pathways deregulated in human cancers, including Cyclin D1, PCNA, Ki-67, LSD1, LOXL2, MT-MMP1, DPAGT1, E-cadherin, OCT4A, and H3K4me1/2. These orthotopic mouse models are likely to be useful tools for gaining insights into the activity and mechanisms of novel oral cancer drug candidates.