Faculty Bibliography

Prostate cancer is a leading cause of cancer death in men in developed countries. While early stage disease can often be cured, many patients eventually develop castration resistant prostate cancer (CRPC). The majority of CRPC patients have bone metastases, which cause significant morbidity and mortality. Although there is no cure for prostate cancer metastatic to bone, several bone-targeted agents have been approved to prevent skeletal-related events (SREs). Among them, bisphosphonates were the first class of drugs investigated for prevention of SREs. Denosumab is a recently approved agent that binds to the receptor activator of nuclear factor-kappaB ligand (RANKL) as a humanized monoclonal antibody. Both agents target prostate cancer skeletal metastasis through the inhibition of bone resorption. Alpharadin is the first radiopharmaceutical agent that has significant overall survival benefit. It has benefits in pain palliation and SREs as well. Another newly approved drug is Abiraterone acetate, which decreases circulating levels of testosterone by targeting an enzyme expressed in the testis and the adrenal, as well as in prostate cancer tissues. This review outlines the clinical and preclinical data supporting the use of these and new agents in development for CRPC with bone metastasis.

ABSTRACT: BACKGROUND: Bacterial infections have been linked to malignancies due to their ability to induce chronic inflammation. We investigated the association of oral bacteria in oral squamous cell carcinoma (OSCC/tumor) tissues and compared with adjacent non-tumor mucosa sampled 5 cm distant from the same patient (n = 10). By using culture-independent 16S rRNA approaches, denaturing gradient gel electrophoresis (DGGE) and cloning and sequencing, we assessed the total bacterial diversity in these clinical samples. RESULTS: DGGE fingerprints showed variations in the band intensity profiles within non-tumor and tumor tissues of the same patient and among the two groups. The clonal analysis indicated that from a total of 1200 sequences characterized, 80 bacterial species/phylotypes were detected representing six phyla, Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, Actinobacteria and uncultivated TM7 in non-tumor and tumor libraries. In combined library, 12 classes, 16 order, 26 families and 40 genera were observed. Bacterial species, Streptococcus sp. oral taxon 058, Peptostreptococcus stomatis, Streptococcus salivarius, Streptococcus gordonii, Gemella haemolysans, Gemella morbillorum, Johnsonella ignava and Streptococcus parasanguinis I were highly associated with tumor site where as Granulicatella adiacens was prevalent at non-tumor site. Streptococcus intermedius was present in 70% of both non-tumor and tumor sites. CONCLUSIONS: The underlying changes in the bacterial diversity in the oral mucosal tissues from non-tumor and tumor sites of OSCC subjects indicated a shift in bacterial colonization. These most prevalent or unique bacterial species/phylotypes present in tumor tissues may be associated with OSCC and needs to be further investigated with a larger sample size.

A number of cancers predominantly metastasize to bone, due to its complex microenvironment and multiple types of constitutive cells. Prostate cancer especially has been shown to localize preferentially to bones with higher marrow cellularity. Using an experimental prostate cancer metastasis model, we investigated the effects of cyclophosphamide, a bone marrow-suppressive chemotherapeutic drug, on the development and growth of metastatic tumors in bone. Priming the murine host with cyclophosphamide before intracardiac tumor cell inoculation was found to significantly promote tumor localization and subsequent growth in bone. Shortly after cyclophosphamide treatment, there was an abrupt expansion of myeloid lineage cells in the bone marrow and the peripheral blood, associated with increases in cytokines with myelogenic potential such as C-C chemokine ligand (CCL)2, interleukin (IL)-6, and VEGF-A. More importantly, neutralizing host-derived murine CCL2, but not IL-6, in the premetastatic murine host significantly reduced the prometastatic effects of cyclophosphamide. Together, our findings suggest that bone marrow perturbation by cytotoxic chemotherapy can contribute to bone metastasis via a transient increase in bone marrow myeloid cells and myelogenic cytokines. These changes can be reversed by inhibition of CCL2.

Williams syndrome is a developmental disorder with a genetic basis, which results in an uneven cognitive profile with relatively strong language skills and severely impaired visuospatial abilities. To better understand the brain structure underlying this profile, we compared individuals with Williams syndrome with controls using multimodal neuroimaging data and new analytic methods (diffeomorphic mapping and atlas-based analysis). People with Williams syndrome had basal ganglia atrophy, while the fusiform, the medium temporal gyri, and the cerebellar cortex were relatively preserved. The right superior longitudinal fasciculus, the left frontooccipital fasciculus, the caudate, and the cingulum demonstrated increased fractional anisotropy, whereas the corticospinal tract revealed decreased values. These findings may be linked to the uneven cognitive profile evident in Williams syndrome.

Bisphosphonates (BPs) are the standard of care for patients with metastatic cancer and multiple myeloma to prevent skeletal complications (e.g., severe bone pain, pathologic fracture, etc.) and to treat osteoporosis. The cause and effect relationship between BPs and BP-related osteonecrosis of the jaws (BRONJ) is not well established. Current research suggests that bacterial biofilms may play a significant role in the pathogenesis of BRONJ. Recently, we have shown that BRONJ lesions are heavily colonized by oral bacteria and present many clinical challenges as they are difficult to culture and antibiotic resistance may result in misguided antibiotic therapy. Here we highlight the crosstalk among the oral bacteria and host immune response in BRONJ subjects. Using 16S rDNA molecular technique we characterize the total bacterial profile of BRONJ, BP and control subjects. Denaturing gradient gel electrophoresis cluster analysis revealed three clusters each representing the three groups, control, BP and BRONJ indicating that the microbiome present in tissue samples was distinct to each group. DGGE band pattern indicated that the BRONJ group had less bacterial diversity as compared to control indicating that high abundance of specific bacteria colonizing the BRONJ lesion. 16S sequencing and clonal analysis showed 6 phyla in all three groups. The phylum Firmicutes was predominant in BRONJ group (72%) followed by BP group (70%) as compared to control group (59%). The Chi-square test also showed significant differences in percent relative distribution of phyla, between control/BP groups (p<0.001), control/BRONJ (p<0.001) and BP/BRONJ (p<0.05). There was significantly increase in the gram positive bacteria in BRONJ group. PCR Array analysis indicated that the host genes responsible for antibacterial response such as MPO, CTSG, and NOD2 were significantly down regulated. Deficient innate immune responses to microorganisms together with poor healing and repair provide continuous opportunities for expanding!

PTH stimulates bone formation and increases hematopoietic stem cells through mechanisms as yet uncertain. The purpose of this study was to identify mechanisms by which PTH links actions on cells of hematopoietic origin with osteoblast-mediated bone formation. C57B6 mice (10 d) were nonlethally irradiated and then administered PTH for 5-20 d. Irradiation reduced bone marrow cellularity with retention of cells lining trabeculae. PTH anabolic activity was greater in irradiated vs. nonirradiated mice, which could not be accounted for by altered osteoblasts directly or osteoclasts but instead via an altered bone marrow microenvironment. Irradiation increased fibroblast growth factor 2, TGFbeta, and IL-6 mRNA levels in the bone marrow in vivo. Irradiation decreased B220 cell numbers, whereas the percent of Lin(-)Sca-1(+)c-kit(+) (LSK), CD11b(+), CD68(+), CD41(+), Lin(-)CD29(+)Sca-1(+) cells, and proliferating CD45(-)Nestin(+) cells was increased. Megakaryocyte numbers were reduced with irradiation and located more closely to trabecular surfaces with irradiation and PTH. Bone marrow TGFbeta was increased in irradiated PTH-treated mice, and inhibition of TGFbeta blocked the PTH augmentation of bone in irradiated mice. In conclusion, irradiation created a permissive environment for anabolic actions of PTH that was TGFbeta dependent but osteoclast independent and suggests that a nonosteoclast source of TGFbeta drives mesenchymal stem cell recruitment to support PTH anabolic actions.

Prostate carcinoma frequently metastasizes to bone where the microenvironment facilitates its growth. Inhibition of bone resorption is effective in reducing tumor burden and bone destruction in prostate cancer. However, whether drugs that inhibit osteoclast function inhibit tumor growth independent of inhibition of bone resorption is unclear. Calcium is released during bone resorption and the calcium sensing receptor is an important regulator of cancer cell proliferation. The goal of this investigation was to elucidate the role of calcium released during bone resorption and to determine the impact of drugs which suppress bone resorption on tumor growth in bone. To compare tumor growth in a skeletal versus non-skeletal site, equal numbers of canine prostate cancer cells expressing luciferase (ACE-1(luc)) were inoculated into a simple collagen matrix, neonatal mouse vertebrae (vossicles), human de-proteinized bone, or a mineralized collagen matrix. Implants were placed subcutaneously into athymic mice. Luciferase activity was used to track tumor growth weekly, and at one month tumors were dissected for histologic analysis. Luciferase activity and tumor size were greater in vossicles, de-proteinized bone and mineralized collagen matrix versus non-mineralized collagen implants. The human osteoblastic prostate carcinoma cell line C4-2b also grew better in a mineral rich environment with a greater proliferation of C4-2b cells reflected by Ki-67 staining. Zoledronic acid (ZA), a bisphosphonate, and recombinant OPG-Fc, a RANKL inhibitor, were administered to mice bearing vertebral implants (vossicles) containing ACE-1 osteoblastic prostate cancer cells. Vossicles or collagen matrices were seeded with ACE-1(luc) cells subcutaneously in athymic mice (2 vossicles, 2 collagen implants/mouse). Mice received ZA (5 mug/mouse, twice/week), (OPG-Fc at 10mg/kg, 3 times/week) or vehicle, and luciferase activity was measured weekly. Histologic analysis of the tumors, vossicles and endogenous bones and serum biochemistry were performed. Antiresorptive administration was associated with decreased serum TRAP5b, reduced osteoclast numbers, and increased tibia and vossicle bone areas. ZA significantly decreased bone marrow calcium concentrations without affecting serum calcium. ZA and OPG-Fc significantly inhibited tumor growth in bone but not in collagen implants. In conclusion, the inhibitory effects of ZA or OPG-Fc on prostate tumor growth in bone are mediated via blocking bone resorption and calcium release from bone.

We have developed a new method to provide a comprehensive quantitative analysis of brain anatomy in cerebral palsy patients, which makes use of two techniques: diffusion tensor imaging and automated 3D whole brain segmentation based on our brain atlas and a nonlinear normalization technique (large-deformation diffeomorphic metric mapping). This method was applied to 13 patients and normal controls. The reliability of the automated segmentation revealed close agreement with the manual segmentation. We illustrate some potential applications for individual characterization and group comparison. This technique also provides a framework for determining the impact of various neuroanatomic features on brain functions.

Prostate cancer cells commonly spread through the circulation, but few successfully generate metastatic foci in bone. Osteoclastic cellular activity has been proposed as an initiating event for skeletal metastasis. Megakaryocytes (MKs) inhibit osteoclastogenesis, which could have an impact on tumor establishment in bone. Given the location of mature MKs at vascular sinusoids, they may be the first cells to physically encounter cancer cells as they enter the bone marrow. Identification of the interaction between MKs and prostate cancer cells was the focus of this study. K562 (human MK precursors) and primary MKs derived from mouse bone marrow hematopoietic precursor cells potently suppressed prostate carcinoma PC-3 cells in coculture. The inhibitory effects were specific to prostate carcinoma cells and were enhanced by direct cell-cell contact. Flow cytometry for propidium iodide (PI) and annexin V supported a proapoptotic role for K562 cells in limiting PC-3 cells. Gene expression analysis revealed reduced mRNA levels for cyclin D1, whereas mRNA levels of apoptosis-associated specklike protein containing a CARD (ASC) and death-associated protein kinase 1 (DAPK1) were increased in PC-3 cells after coculture with K562 cells. Recombinant thrombopoietin (TPO) was used to expand MKs in the marrow and resulted in decreased skeletal lesion development after intracardiac tumor inoculation. These novel findings suggest a potent inhibitory role of MKs in prostate carcinoma cell growth in vitro and in vivo. This new finding, of an interaction of metastatic tumors and hematopoietic cells during tumor colonization in bone, ultimately will lead to improved therapeutic interventions for prostate cancer patients.

Collagen constitutes the most abundant protein in the bone extracellular matrix and triggers receptor mediated cell signaling. The alpha2beta1 integrin is a major collagen receptor on osteoblasts and osteoclasts and previous in vitro studies indicated that collagen-alpha2beta1 is essential for osteoblast differentiation. In osteoclasts, the alphavbeta3 integrin is known to mediate bone resorption; however, the role of alpha2beta1 in osteoclast function is not known. The purpose of this study was to determine the role of alpha2beta1 integrin in osteoclastogenesis and function in vitro and in vivo. Bone marrow derived wild type osteoclasts showed increased cell surface expression of the alpha2 integrin subunit during osteoclast differentiation in vitro as determined by flow cytometry and immunofluorescence. Stimulation of bone marrow derived osteoclasts with soluble type I collagen led to increased tyrosine phosphorylation, including src phosphorylation as detected via western blot analyses. This increased phosphorylation was reduced but not completely inhibited by alpha2beta1 blocking antibodies, suggesting that other collagen receptors may compensate in the absence of alpha2 integrin signaling. Male C57BL/6 mice (10 wks) were examined via micro-computed tomography and bone histomorphometry. The alpha2-/- mice had slight but significantly decreased femur length, diameter, and cortical bone density compared with wild-type littermates. There were no differences in other trabecular or cortical bone parameters including osteoclast number/perimeter evaluated in TRAP stained sections. Interestingly, alpha2-/- osteoclasts had increased in vitro resorptive capacity as measured via resorption pit assay using Osteologic discs. Real time qPCR performed in cultures of differentiating osteoclasts revealed no differences in expression of osteoclast markers including beta3 integrin, calcitonin, cathepsin K, and Nfatc-1. There were no significant differences in serum TRAP levels in alpha2-/- vs. wild type mice.!