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The recent elucidation of the genomic landscape of head and neck squamous cell carcinoma (HNSCC) has provided a unique opportunity to develop selective cancer treatment options. These efforts will require the establishment of relevant HNSCC models for preclinical testing. Here, we performed full exome and transcriptome sequencing of a large panel of HNSCC-derived cells from different anatomical locations and human papillomavirus (HPV) infection status. These cells exhibit typical mutations in TP53, FAT1, CDK2NA, CASP8, and NOTCH1, and copy number variations (CNVs) and mutations in PIK3CA, HRAS, and PTEN that reflect the widespread activation of the PI3K-mTOR pathway. SMAD4 alterations were observed that may explain the decreased tumor suppressive effect of TGF-β in HNSCC. Surprisingly, we identified HPV+ HNSCC cells harboring TP53 mutations, and documented aberrant TP53 expression in a subset of HPV+ HNSCC cases. This analysis also revealed that most HNSCC cells harbor multiple mutations and CNVs in epigenetic modifiers (e.g., EP300, CREBP, MLL1, MLL2, MLL3, KDM6A, and KDM6B) that may contribute to HNSCC initiation and progression. These genetically-defined experimental HNSCC cellular systems, together with the identification of novel actionable molecular targets, may now facilitate the pre-clinical evaluation of emerging therapeutic agents in tumors exhibiting each precise genomic alteration.

Objectives: Exogenous administration of cholecystokinin (CCK) induces hypertrophy and hyperplasia of the pancreas with an increase in DNA content. We hypothesized that endogenous CCK is involved in the malignant progression of pancreatic intraepithelial neoplasia (PanIN) lesions and the fibrosis associated with pancreatic cancer., Methods: The presence of CCK receptors in early PanIN lesions was examined by immunohistochemistry in mouse and human pancreas. Pdx1-Cre/LSL-Kras transgenic mice were randomized to receive either untreated drinking water or water supplemented with a CCK receptor antagonist (proglumide, 0.1 mg/mL). Pancreas from the mice were removed and examined histologically for number and grade of PanINs after 1, 2, or 4 months of antagonist therapy., Results: Both CCK-A and CCK-B receptors were identified in early stage PanINs from mouse and human pancreas. The grade of PanIN lesions was reversed, and progression to advanced lesions arrested in mice treated with proglumide compared with the controls (P = 0.004). Furthermore, pancreatic fibrosis was significantly reduced in antagonist-treated animals compared with vehicle (P < 0.001)., Conclusions: These findings demonstrate that endogenous CCK is in part responsible for the development and progression of pancreatic cancer. The use of CCK receptor antagonists may have a role in cancer prophylaxis in high-risk subjects and may reduce fibrosis in the microenvironment.

We have previously designed and characterized versions of anthrax lethal toxin that are selectively cytotoxic in the tumor microenvironment and which display broad and potent anti-tumor activities in vivo. Here, we have performed the first direct comparison of the safety and efficacy of three engineered anthrax lethal toxin variants requiring activation by either matrix-metalloproteinases (MMPs), urokinase plasminogen activator (uPA) or co-localized MMP/uPA activities. C57BL/6J mice were challenged with six doses of engineered toxins via intraperitoneal (I.P.) or intravenous (I.V.) dose routes to determine the maximum tolerated dose for six administrations (MTD6) and dose-limiting toxicities. Efficacy was evaluated using the B16-BL6 syngraft model of melanoma; mice bearing established tumors were treated with six I.P. doses of toxin and tumor measurements and immunohistochemistry, paired with terminal blood work, were used to elaborate upon the anti-tumor mechanism and relative efficacy of each variant. We found that MMP-, uPA- and dual MMP/uPA-activated anthrax lethal toxins exhibited the same dose-limiting toxicity; dose-dependent GI toxicity. In terms of efficacy, all three toxins significantly reduced primary B16-BL6 tumor burden, ranging from 32% to 87% reduction, and they also delayed disease progression as evidenced by dose-dependent normalization of blood work values. While target organ toxicity and effective doses were similar amongst the variants, the dual MMP/uPA-activated anthrax lethal toxin exhibited the highest I.P. MTD6 and was 1.5-3-fold better tolerated than the single MMP- and uPA-activated toxins. Overall, we demonstrate that this dual MMP/uPA-activated anthrax lethal toxin can be administered safely and is highly effective in a preclinical model of melanoma. This modified bacterial cytotoxin is thus a promising candidate for further clinical development and evaluation for use in treating human cancers., (Published by Elsevier Inc.)

Background: Cetuximab, a monoclonal blocking antibody against the epidermal growth factor receptor EGFR, has been approved for the treatment of squamous cell carcinomas of the head and neck (HNSCC). However, only few patients display long-term responses, prompting the search for cetuximab resistance mechanisms and new therapeutic options enhancing cetuximab effectiveness., Methods: Cetuximab-sensitive HNSCC cells were retro-engineered to express PIK3CA and RAS oncogenes. These cells and HNSCC cells harboring endogenous PIK3CA and RAS oncogenes were xenografted into mice (n = 10 per group) and studied for their biochemical, antitumor, antiangiogenic, and antilymphangiogenic responses to cetuximab and mTOR targeting agents. All P values are two-sided., Results: Cetuximab treatment of PIK3CA- and RAS-expressing HNSCC xenografts promoted an initial antitumor response, but all tumors relapsed within few weeks. In these tumors, cetuximab did not decrease the activity of mTOR, a downstream signaling target of EGFR, PIK3CA, and RAS. The combined administration of cetuximab and mTOR inhibitors exerted a remarkably increased antitumor activity, particularly in HNSCC cells that are resistant to cetuximab as a single agent. Indeed, cotargeting mTOR together with cetuximab caused a rapid tumor collapse of both PIK3CA- and RAS-expressing HNSCC xenografts (P < .001), concomitant with reduced proliferation (P < .001) and lymphangiogenesis (P < .001)., Conclusion: The presence of PIK3CA and RAS mutations and other alterations affecting the mTOR pathway activity in HNSCC could be exploited to predict the potential resistance to cetuximab, and to select the patients that may benefit the most from the concomitant administration of cetuximab and PI3K and/or mTOR inhibitors as a precision molecular therapeutic option for HNSCC patients., (© The Author 2014. Published by Oxford University Press.)

Membrane-anchored serine proteases serve as important regulators of multiple developmental and homoeostatic processes in mammals. TMPRSS13 (transmembrane protease, serine 13; also known as mosaic serine protease large-form, MSPL) is a membrane-anchored serine protease with unknown biological functions. In the present study, we used mice with the Tmprss13 gene disrupted by a β-galactosidase-neomycin fusion gene insertion to study the expression and function of the membrane-anchored serine protease. High levels of Tmprss13 expression were found in the epithelia of the oral cavity, upper digestive tract and skin. Compatible with this expression pattern, Tmprss13-deficient mice displayed abnormal skin development, leading to a compromised barrier function, as measured by the transepidermal fluid loss rate of newborn mice. The present study provides the first biological function for the transmembrane serine protease TMPRSS13.

Mutually exclusive activating mutations in the GNAQ and GNA11 oncogenes, encoding heterotrimeric Gαq family members, have been identified in ∼ 83% and ∼ 6% of uveal and skin melanomas, respectively. However, the molecular events underlying these GNAQ-driven malignancies are not yet defined, thus limiting the ability to develop cancer-targeted therapies. Here, we focused on the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway that controls organ size. We found that Gαq stimulates YAP through a Trio-Rho/Rac signaling circuitry promoting actin polymerization, independently of phospholipase Cβ and the canonical Hippo pathway. Furthermore, we show that Gαq promotes the YAP-dependent growth of uveal melanoma cells, thereby identifying YAP as a suitable therapeutic target in uveal melanoma, a GNAQ/GNA11-initiated human malignancy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Context: Denosumab is a humanized monoclonal antibody to receptor activator of nuclear factor-κB ligand used primarily for postmenopausal osteoporosis and skeletal-related events from metastatic cancer. Its safety in children has not been established., Objective: The objective of the study was to investigate the effects of denosumab treatment on skeletal growth and histology., Design: This was an observational case report with radiological and histopathological analyses., Setting: The study was conducted at a clinical research center., Patients: A 9-year-old boy with fibrous dysplasia treated with a 7-month course of denosumab participated in the study., Intervention: Histological analyses were performed and compared on growth plates from limbs that had been amputated before and 17 months after denosumab treatment., Main Outcome Measures: Skeletal radiographs and bone histopathology from before and after treatment were measured., Results: After initiating denosumab, sclerotic metaphyseal bands appeared on radiographs. Posttreatment radiographs revealed migration of the bands away from the growth plates, consistent with continued linear growth. Histologically, the bands were composed of horizontally arranged trabeculae containing calcified cartilage. This cartilage appeared to derive from unresorbed primary spongiosa as a result of osteoclast inhibition by denosumab, similar to what has been observed with bisphosphonates. By 17 months after treatment, active bone resorption and formation had returned, as evidenced by the presence of active osteoclasts in resorption pits and osteoid surfaces., Conclusions: Further studies are needed to determine the safety of denosumab on the growing skeleton. However, in this child there was continued epiphyseal activity both during and after treatment and reversal of bone turnover suppression after treatment discontinuation, suggesting that denosumab did not have significant adverse effects on growth.

We have recently gained a remarkable understanding of the mutational landscape of head and neck squamous cell carcinoma (HNSCC). However, the nature of the dysregulated signaling networks contributing to HNSCC progression is still poorly defined. Here, we have focused on the role of the family of mitogen activated kinases (MAPKs), extracellular regulated kinase (ERK), c-Jun terminal kinase (JNK) and p38 MAPK in HNSCC. Immunohistochemical analysis of a large collection of human HNSCC tissues revealed that the levels of the phosphorylated active form of ERK1/2 and JNK were elevated in less than 33% and 16% of the cases, respectively. Strikingly, however, high levels of active phospho-p38 were observed in most (79%) of hundreds of tissues analyzed. We explored the biological role of p38 in HNSCC cell lines using three independent approaches: treatment with a specific p38 inhibitor, SB203580; a retro-inhibition strategy consisting in the use of SB203580 combined with the expression of an inhibitor-insensitive mutant form of p38α; and short-hairpin RNAs (shRNAs) targeting p38α. We found that specific blockade of p38 signaling significantly inhibited the proliferation of HNSCC cells both in vitro and in vivo. Indeed, we observed that p38 inhibition in HNSCC cancer cells reduces cancer growth in tumor xenografts and a remarkable decrease in intratumoral blood and lymphatic vessels. We conclude that p38α functions as a positive regulator of HNSCC in the context of the tumor microenvironment, controlling cancer cell growth as well as tumor-induced angiogenesis and lymphangiogenesis., (Published by Elsevier B.V.)

Melanoma represents a significant malignancy in humans and dogs. Different from genetically engineered models, sporadic canine melanocytic neoplasms share several characteristics with human disease that could make dogs a more relevant preclinical model. Canine melanomas rarely arise in sun-exposed sites. Most occur in the oral cavity, with a subset having intra-epithelial malignant melanocytes mimicking the in situ component of human mucosal melanoma. The spectrum of canine melanocytic neoplasia includes benign lesions with some analogy to nevi, as well as invasive primary melanoma, and widespread metastasis. Growing evidence of distinct subtypes in humans, differing in somatic and predisposing germ-line genetic alterations, cell of origin, epidemiology, relationship to ultraviolet radiation and progression from benign to malignant tumors, may also exist in dogs. Canine and human mucosal melanomas appear to harbor BRAF, NRAS, and c-kit mutations uncommonly, compared with human cutaneous melanomas, although both species share AKT and MAPK signaling activation. We conclude that there is significant overlap in the clinical and histopathological features of canine and human mucosal melanomas. This represents opportunity to explore canine oral cavity melanoma as a preclinical model., (© 2013 The Authors. Pigment Cell & Melanoma Research published by John Wiley & Sons Ltd.)

Human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) incidence is increasing at a near epidemic rate. We investigated whether the mammalian (or mechanistic) target of rapamycin (mTOR) inhibitor, rapamycin, can be used as a concurrent agent to standard-of-care cisplatin/radiation therapy (CRT) to attenuate tumor lactate production, thus enhancing CRT-induced immune-mediated clearance of this antigenic tumor type. A C57Bl/6-derived mouse oropharyngeal epithelial cell line retrovirally transduced with HPV type 16 E6/E7 and human squamous cell carcinoma cell lines were evaluated for their response to rapamycin in vitro with proliferation assays, Western blots, and lactate assays. Clonogenic assays and a preclinical mouse model were used to assess rapamycin as a concurrent agent to CRT. The potential of rapamycin to enhance immune response through lactate attenuation was assessed using quantitative tumor lactate bioluminescence and assessment of cell-mediated immunity using E6/E7-vaccinated mouse splenocytes. Rapamycin alone inhibited mTOR signaling of all cancer cell lines tested in vitro and in vivo. Furthermore, rapamycin administered alone significantly prolonged survival in vivo but did not result in any long-term cures. Given concurrently, CRT/rapamycin significantly enhanced direct cell killing in clonogenic assays and prolonged survival in immunocompromised mice. However, in immunocompetent mice, concurrent CRT/rapamycin increased long-term cures by 21%. Preliminary findings suggest that improved survival involves increased cell killing and enhanced immune-mediated clearance in part due to decreased lactate production. The results may provide rationale for the clinical evaluation of mTOR inhibitors concurrent with standard-of-care CRT for treatment of HPV-positive HNSCC.

Neuroendocrine (NE) differentiation has gained increased attention as a prostate cancer (PC) prognostic marker. The aim of this study is to determine whether host germline genetic variation influences tumor progression and metastasis in C57BL/6-Tg(TRAMP)8247Ng/J (TRAMP) mouse model of aggressive NEPC. TRAMP mice were crossed to the eight progenitor strains of the Collaborative Cross recombinant inbred panel to address this. Tumor growth and metastasis burden were quantified in heterozygous transgene positive F1 male mice at 30 weeks of age. Compared to wild-type C57BL/6J-Tg(TRAMP)824Ng/J males, TRAMP x CAST/EiJ, TRAMP x NOD/ShiLtJ and TRAMP x NZO/HlLtJ F1 males displayed significant increases in tumor growth. Conversely, TRAMP x WSB/EiJ and TRAMP x PWK/PhJ F1 males displayed significant reductions in tumor growth. Interestingly, despite reduced tumor burden, TRAMP x WSB/EiJ males had an increased nodal metastasis burden. Patterns of distant pulmonary metastasis tended to follow the same patterns as that of local dissemination in each of the strains. All tumors and metastases displayed positive staining for NE markers, synaptophysin, and FOXA2. These experiments conclusively demonstrate that the introduction of germline variation by breeding modulates tumor growth, local metastasis burden, and distant metastasis frequency in this model of NEPC. These strains will be useful as model systems to facilitate the identification of germline modifier genes that promote the development of aggressive forms of PC.

Objectives: The diagnosis of cervical lymph node metastasis in head and neck squamous cell carcinoma (HNSCC) patients constitutes an essential requirement for clinical staging and treatment selection. However, clinical assessment by physical examination and different imaging modalities, as well as by histological examination of routine lymph node cryosections can miss micrometastases, while false positives may lead to unnecessary elective lymph node neck resections. Here, we explored the feasibility of developing a sensitive assay system for desmoglein 3 (DSG3) as a predictive biomarker for lymph node metastasis in HNSCC., Materials and Methods: DSG3 expression was determined in multiple general cancer- and HNSCC-tissue microarrays (TMAs), in negative and positive HNSCC metastatic cervical lymph nodes, and in a variety of HNSCC and control cell lines. A nanostructured immunoarray system was developed for the ultrasensitive detection of DSG3 in lymph node tissue lysates., Results: We demonstrate that DSG3 is highly expressed in all HNSCC lesions and their metastatic cervical lymph nodes, but absent in non-invaded lymph nodes. We show that DSG3 can be rapidly detected with high sensitivity using a simple microfluidic immunoarray platform, even in human tissue sections including very few HNSCC invading cells, hence distinguishing between positive and negative lymph nodes., Conclusion: We provide a proof of principle supporting that ultrasensitive nanostructured assay systems for DSG3 can be exploited to detect micrometastatic HNSCC lesions in lymph nodes, which can improve the diagnosis and guide in the selection of appropriate therapeutic intervention modalities for HNSCC patients., (Published by Elsevier Ltd.)

The expression of heme oxygenase-1 (HO-1) was shown to be increased in multiple tumors compared with their surrounding healthy tissues and was also observed to be up-regulated in oral squamous cell carcinomas (OSCC). However, conflicting results were obtained and little information is available regarding HO-1 significance in head and neck squamous cell carcinoma (HNSCC). Therefore, the aim of the present study was to perform a wide screening of HO-1 expression in a large collection of human primary HNSCCs and to correlate the results with clinical and pathological parameters. For this purpose, we investigated the expression of this protein by immunohistochemistry (IHC) in tissue microarrays (TMAs) of HNSCC and in an independent cohort of paraffin-embedded tumor specimens. HO-1 expression was further validated by real-time qPCR performed on selected laser capture-microdissected (LCM) oral tissue samples. Both the number of HO-1-positive samples and HO-1 immunoreactivity in the cancerous tissues were significantly higher than those in the non-tumor tissues. These results were confirmed at the mRNA level. Interestingly, HO-1 localization was observed in the nucleus, and the rate of nuclear HO-1 in HNSCC was higher than that in non-malignant tissues. Nuclear HO-1 was observed in HNSCC cell lines and increased even further following hemin treatment. Analysis of HO-1 expression and sub-cellular localization in a mouse model of squamous cell carcinoma (SCC) and in human HNSCC revealed that nuclear HO-1 increases with tumor progression. Taken together, these results demonstrate that HO-1 is up-regulated in HNSCC and that nuclear localization of HO-1 is associated with malignant progression in this tumor type., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The integrity of the epidermis and mucosal epithelia is highly dependent on resident self-renewing stem cells, which makes them vulnerable to physical and chemical insults compromising the repopulating capacity of the epithelial stem cell compartment. This is frequently the case in cancer patients receiving radiation or chemotherapy, many of whom develop mucositis, a debilitating condition involving painful and deep mucosal ulcerations. Here, we show that inhibiting the mammalian target of rapamycin (mTOR) with rapamycin increases the clonogenic capacity of primary human oral keratinocytes and their resident self-renewing cells by preventing stem cell senescence. This protective effect of rapamycin is mediated by the increase in expression of mitochondrial superoxide dismutase (MnSOD), and the consequent inhibition of ROS formation and oxidative stress. mTOR inhibition also protects from the loss of proliferative basal epithelial stem cells upon ionizing radiation in vivo, thereby preserving the integrity of the oral mucosa and protecting from radiation-induced mucositis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Head and neck cancer is a devastating disease that afflicts many individuals worldwide. Conventional therapies are successful in only a limited subgroup and often leave the patient with disfigurement and long lasting adverse effects on normal physiologic functions. The field is in dire need of new therapies. Oncolytic viral as well as targeted therapies have shown some success in other malignancies and are attractive for the treatment of head and neck cancer. Recently, it has been shown that a subset of head and neck cancers is human papillomavirus (HPV) positive and that this subset of cancers is biologically distinct and more sensitive to chemoradiation therapies although the underlying mechanism is unclear. However, chemoresistance remains a general problem. One candidate mediator of therapeutic response, which is of interest for the targeting of both HPV-positive and -negative tumors is the human DEK proto-oncogene. DEK is upregulated in numerous tumors including head and neck cancers regardless of their HPV status. Depletion of DEK in tumor cells in culture results in sensitivity to genotoxic agents, particularly in rapidly proliferating cells. This suggests that tumors with high DEK protein expression may be correlated with poor clinical response to clastogenic therapies. Targeting molecules such as DEK in combination with new and/or conventional therapies, holds promise for novel future therapeutics for head and neck cancer., (Copyright © 2012 Mosby, Inc. All rights reserved.)

Colitis-associated colorectal cancers are an etiologically distinct subgroup of colon cancers that occur in individuals suffering from inflammatory bowel disease and arise as a consequence of persistent exposure of hyperproliferative epithelial stem cells to an inflammatory microenvironment. An intrinsic defect in the intestinal epithelial barrier has been proposed to be one of several factors that contribute to the inappropriate immune response to the commensal microbiota that underlies inflammatory bowel disease. Matriptase is a membrane-anchored serine protease encoded by Suppression of Tumorigenicity-14 (ST14) that strengthens the intestinal epithelial barrier by promoting tight junction formation. Here, we show that intestinal epithelial-specific ablation of St14 in mice causes formation of colon adenocarcinoma with very early onset and high penetrance. Neoplastic progression is preceded by a chronic inflammation of the colon that resembles human inflammatory bowel disease and is promoted by the commensal microbiota. This study demonstrates that inflammation-associated colon carcinogenesis can be initiated and promoted solely by an intrinsic intestinal permeability barrier perturbation, establishes St14 as a critical tumor-suppressor gene in the mouse gastrointestinal tract and adds matriptase to the expanding list of pericellular proteases with tumor-suppressive functions.

Fibrous dysplasia (FD) is a skeletal disease caused by somatic activating mutations of the cyclic adenosine monophosphate (cAMP)-regulating protein, α-subunit of the Gs stimulatory protein (G(s) α). These mutations lead to replacement of normal bone by proliferative osteogenic precursors, resulting in deformity, fracture, and pain. Medical treatment has been ineffective in altering the disease course. Receptor activator of NF-κB ligand (RANKL) is a cell-surface protein involved in many cellular processes, including osteoclastogenesis, and is reported to be overexpressed in FD-like bone cells. Denosumab is a humanized monoclonal antibody to RANKL approved for treatment of osteoporosis and prevention of skeletal-related events from bone metastases. We present the case of a 9-year-old boy with severe FD who was treated with denosumab for a rapidly expanding femoral lesion. Immunohistochemical staining on a pretreatment bone biopsy specimen revealed marked RANKL expression. He was started on monthly denosumab, with an initial starting dose of 1 mg/kg and planned 0.25 mg/kg dose escalations every 3 months. Over 7 months of treatment he showed marked reduction in pain, bone turnover markers (BTMs), and tumor growth rate. Denosumab did not appear to impair healing of a femoral fracture that occurred while on treatment. With initiation of treatment he developed hypophosphatemia and secondary hyperparathyroidism, necessitating supplementation with phosphorus, calcium, and calcitriol. BTMs showed rapid and sustained suppression. With discontinuation there was rapid and dramatic rebound of BTMs with cross-linked C-telopeptide (reflecting osteoclast activity) exceeding pretreatment levels, accompanied by severe hypercalcemia. In this child, denosumab lead to dramatic reduction of FD expansion and FD-related bone pain. Denosumab was associated with clinically significant disturbances of mineral metabolism both while on treatment and after discontinuation. Denosumab treatment of FD warrants further study to confirm efficacy and determine potential morbidity, as well as to determine the mechanism of RANKL in the pathogenesis of FD and related bone marrow stromal cell diseases., (Copyright © 2012 American Society for Bone and Mineral Research.)

Capillary morphogenesis protein-2 (CMG2) functions as an anthrax toxin receptor that plays an essential role in anthrax pathogenesis. Although mutations in CMG2 have been identified to cause two human autosomal recessive disorders, Juvenile Hyaline Fibromatosis and Infantile Systemic Hyalinosis, both characterized by excess hyaline material deposition in connective tissues, the physiologic function of CMG2 remains elusive. To study the roles of CMG2 in normal physiology, here we performed detailed histological analyses of the CMG2-null mice we generated previously. While no morphological or histological defects were observed in CMG2(-/-) male mice, CMG2(-/-) female mice were unable to produce any offspring due to a defect in parturition. We found that deletion of CMG2 resulted in a diffuse deposition of collagen within the myometrium of CMG2(-/-) females, causing remarkable morphological changes to their uteri. This collagen accumulation also led to loss of smooth muscle cells in the myometrium of CMG2(-/-) mice, apparently disabling uterine contractile function during parturition. As a consequence, even though pregnant CMG2(-/-) mice were able to carry the gestation to full term, they were unable to deliver pups. However, the fully-developed fetuses could be successfully delivered by Cesarean section and survived to adulthood when fostered. Our results demonstrate that CMG2 is not required for normal mouse embryonic development but is indispensable for murine parturition. In parallel to its role in anthrax toxin binding and internalization, herein we provide evidence that CMG2 may function as a collagen receptor which is essential for maintaining collagen homeostasis in the uterus., (Published by Elsevier Inc.)

Fibroblast growth factor receptors (FGFRs) are tyrosine kinase receptors which have been implicated in breast cancer. The aim of this study was to evaluate FGFR-1, -2, -3, and -4 protein expressions in normal murine mammary gland development, and in murine and human breast carcinomas. Using immunohistochemistry and Western blot, we report a hormonal regulation of FGFR during postnatal mammary gland development. Progestin treatment of adult virgin mammary glands resulted in changes in localization of FGFR-3 from the cytoplasm to the nucleus, while treatment with 17-β-estradiol induced changes in the expressions and/or localizations of FGFR-2 and -3. In murine mammary carcinomas showing different degrees of hormone dependence, we found progestin-induced increased expressions, mainly of FGFR-2 and -3. These receptors were constitutively activated in hormone-independent variants. We studied three luminal human breast cancer cell lines growing as xenografts, which particularly expressed FGFR-2 and -3, suggesting a correlation between hormonal status and FGFR expression. Most importantly, in breast cancer samples from 58 patients, we found a strong association (P < 0.01; Spearman correlation) between FGFR-2 and -3 expressions and a weaker correlation of each receptor with estrogen receptor expression. FGFR-4 correlated with c-erbB2 over expression. We conclude that FGFR-2 and -3 may be mechanistically linked and can be potential targets for treatment of estrogen receptor-positive breast cancer patients.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide. It is accepted that breast cancer is not a single disease, but instead constitutes a spectrum of tumor subtypes with distinct cellular origins, somatic changes, and etiologies. Molecular gene expression studies have divided breast cancer into several categories, i.e. basal-like, ErbB2 enriched, normal breast-like (adipose tissue gene signature), luminal subtype A, luminal subtype B, and claudin-low. Chances are that as our knowledge increases, each of these types will also be subclassified. More than 66% of breast carcinomas express estrogen receptor alpha (ERα) and respond to antiestrogen therapies. Most of these ER+ tumors also express progesterone receptors (PRs), the expression of which has been considered as a reliable marker of a functional ER. In this paper we will review the evidence suggesting that PRs are valid targets for breast cancer therapy. Experimental data suggest that both PR isoforms (A and B) have different roles in breast cancer cell growth, and antiprogestins have already been clinically used in patients who have failed to other therapies. We hypothesize that antiprogestin therapy may be suitable for patients with high levels of PR-A. This paper will go over the experimental evidence of our laboratory and others supporting the use of antiprogestins in selected breast cancer patients.

Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth, but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) are required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear colocalization of activated ERα with PR. Treatment with the pure antiestrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies showed that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear colocalization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation., (©2012 AACR)

Purpose: The incidence of head and neck squamous cell carcinomas (HNSCC) associated with human papillomavirus (HPV) infection has increased over the past decades in the United States. We aimed at examining the global impact of HPV-associated HNSCC and whether the established key role of mTOR activation in HNSCC is also observed in HPV(+) HNSCC lesions, thereby providing novel treatment options for HPV-associated HNSCC patients., Experimental Design: An international HNSCC tissue microarray (TMA) was used to analyze the expression of p16(INK4A), a surrogate for HPV infection, and Akt-mTOR pathway activation. Results were confirmed in a large collection of HPV(-) and HPV(+) HNSCC cases and in a cervical cancer (CCSCC) TMA. Observations were validated in HNSCC and CCSCC-derived cell lines, which were xenografted into immunodeficient mice for tumorigenesis assays., Results: Approximately 20% of all HNSCC lesions could be classified as HPV(+), irrespective of their country of origin. mTOR pathway activation was observed in most HPV(+) HNSCC and CCSCC lesions and cell lines. The preclinical efficacy of mTOR inhibition by rapamycin and RAD001 was explored in HPV(+) HNSCC and CCSCC tumor xenografts. Both mTOR inhibitors effectively decreased mTOR activity in vivo and caused a remarkable decrease in tumor burden. These results emphasize the emerging global impact of HPV-related HNSCCs and indicate that the activation of the mTOR pathway is a widespread event in both HPV(-) and HPV-associated HNSCC and CCSCC lesions., Conclusions: The emerging results may provide a rationale for the clinical evaluation of mTOR inhibitors as a molecular targeted approach for the treatment of HPV-associated malignancies., (©2012 AACR.)

Head and neck squamous cell carcinoma (HNSCC) is a major public health concern. The recent identification of the mTOR complex 1 (mTORC1) signaling pathway as a highly prevalent molecular signature underlying HNSCC pathogenesis has provided the foundation to search for novel therapeutic approaches to prevent and treat HNSCC. Here, we asked whether metformin, the most widely used medication for the treatment of type II diabetes, which acts in part by stimulating the AMP-activated protein kinase (AMPK) signaling pathway thereby reducing mTORC1 activity, may lower the risk of HNSCC development. Indeed, we show that metformin reduces the growth of HNSCC cells and diminishes their mTORC1 activity by both AMPK-dependent and -independent mechanisms. We also optimized an oral-specific carcinogenesis mouse model that results in the accumulation of multiple oral premalignant lesions at the end of the carcinogen exposure, some of which then spontaneously progress into HNSCC. Using this mouse model, we observed that metformin specifically inhibits mTORC1 in the basal proliferating epithelial layer of oral premalignant lesions. Remarkably, metformin prevented the development of HNSCC by reducing significantly the size and number of carcinogen-induced oral tumoral lesions and by preventing their spontaneous conversion to squamous cell carcinomas. Collectively, our data underscore the potential clinical benefits of using metformin as a targeted chemopreventive agent in the control of HNSCC development and progression., (2012 AACR)

In C4-HD murine mammary carcinomas and in human breast cancer T47D cells, we showed that medroxyprogesterone acetate (MPA) induces a nuclear physical association between estrogen receptor alpha (ERa) and progesterone receptors (PR). The blockade of ERa inhibits cell proliferation mediated by progestins. We hypothesized that this nuclear association between ERa/PR is necessary to trigger progestin-induced cell proliferation and tumor growth. We demonstrated that fulvestrant (FUL, ICI182.780) induced complete regression of C4-HD tumors growing with progestins. MPA treatment induced an early increase in both CCND1 and MYC expression in T47D cells. The blockade of ERa prevented the MPA-dependent transcription of both genes. Specific binding of PR/ERa was observed at the same MPA-sensitive regions at the CCND1 and MYC gene promoters after chromatin immunoprecipitation (ChIP) analysis. ICI inhibited binding of ERa to both gene regulatory sequences while PR binding was unaffected. The nuclear colocalization between both receptors in T47D cells was confirmed by: confocal microscopy, Duolink assays and co-immunoprecipitation assays. In breast cancer samples we also observed a nuclear interaction between both steroid receptors. Our results indicate that the presence of ERa interacting with activated PR at the CCND1 and MYC promoters is required to trigger progestin-induced gene transcription and cell proliferation in breast cancer cells.

Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogues, as part of a molecular-targeted metastasis preventive strategy for the treatment of patients with HNSCC., (©2011 AACR)

Tumor cells can co-opt the promigratory activity of chemokines and their cognate G protein-coupled receptors (GPCRs) to metastasize to regional lymph nodes or distant organs. Indeed, the migration toward SDF-1 (stromal cell-derived factor 1) of tumor cells bearing CXCR4 [chemokine (C-X-C motif) receptor 4] has been implicated in the lymphatic and organ-specific metastasis of various human malignancies. Here, we used chimeric G proteins and GPCRs activated solely by artificial ligands to selectively activate the signaling pathways downstream of specific G proteins and showed that CXCR4-mediated chemotaxis and transendothelial migration of metastatic basal-like breast cancer cells required activation of Gα(13), a member of the Gα(12/13) G protein family, and of the small guanosine triphosphatase Rho. Multiple complementary experimental strategies, including synthetic biology approaches, indicated that signaling-selective inhibition of the CXCR4-Gα(13)-Rho axis prevents the metastatic spread of basal-like breast cancer cells.

Angioproliferative tumors induced by the Kaposi's sarcoma-associated herpesvirus (KSHV) have been successfully treated with rapamycin, which provided direct evidence of the clinical activity of mTOR inhibitors in human malignancies. However, prolonged mTOR inhibition may raise concerns in immunocompromised patients, including AIDS-Kaposi's sarcoma (KS). Here, we explored whether KSHV oncogenes deploy cell type-specific signaling pathways activating mTOR, which could be exploited to halt KS development while minimizing immune suppressive effects. We found that PI3Kγ, a PI3K isoform exhibiting restricted tissue distribution, is strictly required for signaling from the KSHV-encoded vGPCR oncogene to Akt/mTOR. Indeed, by using an endothelial-specific gene delivery system modeling KS development, we provide genetic and pharmacological evidence that PI3Kγ may represent a suitable molecular target for therapeutic intervention in KS., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Tumor-induced osteomalacia (TIO) is a rare and fascinating paraneoplastic syndrome in which patients present with bone pain, fractures, and muscle weakness. The cause is high blood levels of the recently identified phosphate and vitamin D-regulating hormone, fibroblast growth factor 23 (FGF23). In TIO, FGF23 is secreted by mesenchymal tumors that are usually benign, but are typically very small and difficult to locate. FGF23 acts primarily at the renal tubule and impairs phosphate reabsorption and 1α-hydroxylation of 25-hydroxyvitamin D, leading to hypophosphatemia and low levels of 1,25-dihydroxy vitamin D. A step-wise approach utilizing functional imaging (F-18 fluorodeoxyglucose positron emission tomography and octreotide scintigraphy) followed by anatomical imaging (computed tomography and/or magnetic resonance imaging), and, if needed, selective venous sampling with measurement of FGF23 is usually successful in locating the tumors. For tumors that cannot be located, medical treatment with phosphate supplements and active vitamin D (calcitriol or alphacalcidiol) is usually successful; however, the medical regimen can be cumbersome and associated with complications. This review summarizes the current understanding of the pathophysiology of the disease and provides guidance in evaluating and treating these patients. Novel imaging modalities and medical treatments, which hold promise for the future, are also reviewed.

The progression and negative outcome of a variety of human carcinomas are intimately associated with aberrant activity of the c-Met oncogene. The underlying cause of this dysregulation, however, remains a subject of discussion, as the majority of cancer patients do not present with activating mutations in c-Met receptor itself. In this study, we show that the oncogenic protease matriptase is ubiquitously co-expressed with the c-Met in human squamous cell carcinomas and amplifies migratory and proliferative responses of primary epithelial cells to the cognate ligand for c-Met, pro-hepatocyte growth factor/scatter factor (proHGF/SF), through c-Met and Gab1 signaling. Furthermore, the selective genetic ablation of c-Met from matriptase-expressing keratinocytes completely negates the oncogenic potential of matriptase. In addition, matriptase-dependent carcinoma formation could be blocked by the pharmacological inhibition of the Akt-mammalian target of Rapamycin (mTor) pathway. Our data identify matriptase as an initiator of c-Met-Akt-mTor-dependent signaling axis in tumors and reveal mTor activation as an essential component of matriptase/c-Met-induced carcinogenesis. The study provides a specific example of how epithelial transformation can be promoted by epigenetic acquisition of the capacity to convert a widely available paracrine growth factor precursor to its signaling competent state.

In this article, we demonstrate the expression of functional progesterone binding sites at the cell membrane in murine mammary carcinomas that are stimulated by progestins and inhibited by antiprogestins. Using confocal immunofluorescence, ligand binding and cell compartment-specific western blots, we were able to identify the presence of the classical progesterone receptors. Medroxyprogesterone acetate (MPA) and RU-486 (1 × 10(-11) and 1 × 10(-8) M) behaved as agonists activating extracellular signal-regulated kinases (ERKs) and progestin-regulated proteins, except for Cyclin D1 and Tissue factor which failed to increase with 1 × 10(-8) M RU-486, an experimental condition that allows PR to bind DNA. These results predicted a full agonist effect at low concentrations of RU-486. Accordingly, at concentrations lower than 1 × 10(-11) M, RU-486 increased cell proliferation in vitro. This effect was abolished by incubation with the ERK kinase inhibitor PD 98059 or by OH-tamoxifen. In vivo, at a daily dose of 1.2 μg/kg body weight RU-486 increased tumor growth, whereas at 12 mg/kg induces tumor regression. Our results indicate that low concentrations of MPA and RU-486 induce similar agonistic non-genomic effects, whereas RU-486 at higher concentrations may inhibit cell proliferation by genomic-induced effects. This suggests that RU-486 should be therapeutically administered at doses high enough to guarantee its genomic inhibitory effect.

The human oral squamous epithelium plays an important role in maintaining a barrier function against mechanical, physical, and pathological injury. However, the self-renewing cells residing on the basement membrane of the epithelium can give rise to oral squamous cell carcinomas (OSCC), now the sixth most common cancer in the developed world, which is still associated with poor prognosis. This is due, in part, to the limited availability of well-defined culture systems for studying oral epithelial cell biology, which could advance our understanding of the molecular basis of OSCC. Here, we describe methods to successfully isolate large cultures of human oral epithelial cells and fibroblasts from small pieces of donor tissues for use in techniques such as three-dimensional cultures and animal grafts to validate genes suspected of playing a role in OSCC development and progression. Finally, the use of isolated oral epithelial cells in generating iPS cells is discussed which holds promise in the field of oral regenerative medicine.

Genome mining at the turn of the millennium uncovered a new family of type II transmembrane serine proteases (TTSPs) that comprises 17 members in humans and 19 in mice. TTSPs phylogenetically belong to one of four subfamilies: matriptase, hepsin/TMPRSS, corin and HAT/DESC. Whereas a wealth of information now has been gathered as to the physiological functions of members of the hepsin/TMPRSS, matriptase, and corin subfamilies of TTSPs, comparatively little is known about the functions of the HAT/DESC subfamily of proteases. Here we perform a combined expression and functional analysis of this TTSP subfamily. We show that the five human and seven murine HAT/DESC proteases are coordinately expressed, suggesting a level of functional redundancy. We also perform a comprehensive phenotypic analysis of mice deficient in two of the most widely expressed HAT/DESC proteases, TMPRSS11A and HAT, and show that the two proteases are dispensable for development, health, and long-term survival in the absence of external challenges or additional genetic deficits. Our comprehensive expression analysis and generation of TMPRSS11A- and HAT-deficient mutant mouse strains provide a valuable resource for the scientific community for further exploration of the HAT/DESC subfamily proteases in physiological and pathological processes.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although considerable progress has been made in elucidating the etiology of the disease, the prognosis for individuals diagnosed with HNSCC remains poor, underscoring the need for development of additional treatment modalities. HNSCC is characterized by the upregulation of a large number of proteolytic enzymes, including urokinase plasminogen activator (uPA) and an assortment of matrix metalloproteinases (MMPs) that may be expressed by tumor cells, by tumor-supporting stromal cells or by both. Here we explored the use of an intercomplementing anthrax toxin that requires combined cell surface uPA and MMP activities for cellular intoxication and specifically targets the ERK/MAPK pathway for the treatment of HNSCC. We found that this toxin displayed strong systemic anti-tumor activity towards a variety of xenografted human HNSCC cell lines by inducing apoptotic and necrotic tumor cell death, and by impairing tumor cell proliferation and angiogenesis. Interestingly, the human HNSCC cell lines were insensitive to the intercomplementing toxin when cultured ex vivo, suggesting that either the toxin targets the tumor-supporting stromal cell compartment or that the tumor cell requirement for ERK/MAPK signaling differs in vivo and ex vivo. This intercomplementing toxin warrants further investigation as an anti-HNSCC agent.

The overexpression of cyclooxygenase (COX)-2 is a frequent event in squamous cell carcinomas of the head and neck (HNSCC), and non-steroidal anti-inflammatory drugs, which are potent inhibitors of COX-1 and COX-2, exert chemopreventive effects on HNSCC cancer development. COX-2 promotes the release of the pro-inflammatory mediator prostaglandin E2 (PGE2), which acts on its cell surface G protein-coupled receptors EP1, EP2, EP3, and EP4. Here, we investigated the role of PGE2 and its receptors in cellular proliferation in HNSCC. The expression of COX-2 and EP1-4 was examined in immortalized oral epithelial cells and in a representative panel of HNSCC cell lines, and based on these data EP1-EP3 and COX-2 expression were evaluated by immunohistochemistry in a large clinical sample collection using HNSCC tissue microarrays. The ability of selective COX-2 inhibition to block PGE2 secretion was measured by ELISA specific assays. The effects of PGE2 on cell proliferation were evaluated using PGE2, its stable analog, and EP2 and EP3-specific synthetic agonists. The results presented here show that HNSCC tumoral lesions and their derived cell lines constitutively express COX-2 and the EP1, EP2 and EP3 receptors for PGE2. HNSCC cells secrete PGE2, which can be suppressed by low concentrations of COX-2 selective inhibitors, without inhibiting cell proliferation. Exogenously added stable PGE2 and EP3-specific agonists induce DNA synthesis in all HNSCC cell lines tested. Overall, our study supports the emerging notion that PGE2 produced in the tumor microenvironment by the overexpression of COX-2 in tumoral and inflammatory cells may promote the growth of HNSCC cells in an autocrine and paracrine fashion by acting on PGE2 receptors that are widely expressed in most HNSCC cancer cells. In particular, our findings suggest that EP3 receptor may play a more prominent role in HNSCC cell growth promotion, thus providing a rationale for the future evaluation of this PGE2 receptor as a target for HNSCC prevention strategies., (Published by Elsevier Ltd.)

Aims: To study the distribution and clearance of polyethylene glycol (PEG)-ylated single-walled carbon nanotube (SWCNTs) as drug delivery vehicles for the anticancer drug cisplatin in mice., Materials & Methods: PEG layers were attached to SWCNTs and dispersed in aqueous media and characterized using dynamic light scattering, scanning transmission electron microscopy and Raman spectroscopy. Cytotoxicity was assessed in vitro using Annexin-V assay, and the distribution and clearance pathways in mice were studied by histological staining and Raman spectroscopy. Efficacy of PEG-SWCNT-cisplatin for tumor growth inhibition was studied in mice., Results & Discussion: PEG-SWCNTs were efficiently dispersed in aqueous media compared with controls, and did not induce apoptosis in vitro. Hematoxylin and eosin staining, and Raman bands for SWCNTs in tissues from several vital organs from mice injected intravenously with nanotube bioconjugates revealed that control SWCNTs were lodged in lung tissue as large aggregates compared with the PEG-SWCNTs, which showed little or no accumulation. Characteristic SWCNT Raman bands in feces revealed the presence of bilary or renal excretion routes. Attachment of cisplatin on bioconjugates was visualized with Z-contrast scanning transmission electron microscopy. PEG-SWCNT-cisplatin with the attached targeting ligand EGF successfully inhibited growth of head and neck tumor xenografts in mice., Conclusions: PEG-SWCNTs, as opposed to control SWCNTs, form more highly dispersed delivery vehicles that, when loaded with both cisplatin and EGF, inhibit growth of squamous cell tumors.

Background: Increasing evidence has underscored the role of carcinoma associated fibroblasts (CAF) in tumor growth. However, there are controversial data regarding the persistence of inoculated CAF within the tumors. We have developed a model in which murine metastatic ductal mammary carcinomas expressing estrogen and progesterone receptors transit through different stages of hormone dependency. Hormone dependent (HD) tumors grow only in the presence of progestins, whereas hormone independent (HI) variants grow without hormone supply. We demonstrated previously that CAF from HI tumors (CAF-HI) express high levels of FGF-2 and that FGF-2 induced HD tumor growth in vivo. Our main goal was to investigate whether inoculated CAF-HI combined with purified epithelial (EPI) HD cells can induce HD tumor growth., Methods: Purified EPI cells of HD and HI tumors were inoculated alone, or together with CAF-HI, into female BALB/c mice and tumor growth was evaluated. In another set of experiments, purified EPI-HI alone or combined with CAF-HI or CAF-HI-GFP were inoculated into BALB/c or BALB/c-GFP mice. We assessed whether inoculated CAF-HI persisted within the tumors by analyzing inoculated or host CAF in frozen sections of tumors growing in BALB/c or BALB/c-GFP mice. The same model was used to evaluate early stages of tumor development and animals were euthanized at 2, 7, 12 and 17 days after EPI-HI or EPI-HI+CAF-HI inoculation. In angiogenesis studies, tumor vessels were quantified 5 days after intradermal inoculation., Results: We found that admixed CAF-HI failed to induce epithelial HD tumor growth, but instead, enhanced HI tumor growth (p < 0.001). Moreover, inoculated CAF-HI did not persist within the tumors. Immunofluorescence studies showed that inoculated CAF-HI disappeared after 13 days. We studied the mechanisms by which CAF-HI increased HI tumor growth, and found a significant increase in angiogenesis (p < 0.05) in the co-injected mice at early time points., Conclusions: Inoculated CAF-HI do not persist within the tumor mass although they play a role during the first stages of tumor formation promoting angiogenesis. This angiogenic environment is unable to replace the hormone requirement of HD tumors that still need the hormone to recruit the stroma from the host.

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

A mutational analysis of the matrix metalloproteinase (MMP) gene family in human melanoma identified somatic mutations in 23% of melanomas. Five mutations in one of the most commonly mutated genes, MMP8, reduced MMP enzyme activity. Expression of wild-type but not mutant MMP8 in human melanoma cells inhibited growth on soft agar in vitro and tumor formation in vivo, suggesting that wild-type MMP-8 has the ability to inhibit melanoma progression.

Multiple genetic and epigenetic events, including the aberrant expression and function of molecules regulating cell signaling, growth, survival, motility, angiogenesis, and cell cycle control, underlie the progressive acquisition of a malignant phenotype in squamous carcinomas of the head and neck (HNSCC). In this regard, there has been a recent explosion in our understanding on how extracellular components, cell surface molecules, and a myriad of intracellular proteins and second messenger systems interact with each other, and are organized in pathways and networks to control cellular and tissue functions and cell fate decisions. This emerging ability to understand the basic mechanism controlling inter- and intra-cellular communication has provided an unprecedented opportunity to understand how their dysregulation contributes to the growth and dissemination of human cancers. Here, we will discuss the emerging information on how the use of modern technologies, including gene array and proteomic studies, combined with the molecular dissection of aberrant signaling networks, including the EGFR, ras, NFkappaB, Stat, Wnt/beta-catenin, TGF-beta, and PI3K-AKT-mTOR signaling pathways, can help elucidate the molecular mechanisms underlying HNSCC progression. Ultimately, we can envision that this knowledge may provide tremendous opportunities for the diagnosis of premalignant squamous lesions, and for the development of novel molecular-targeted strategies for the prevention and treatment of HNSCC.

The increased molecular understanding of cancerous growth may now afford the opportunity to develop novel therapies targeting specific dysregulated molecular mechanisms contributing to the progression of each cancer type. In this regard, the aberrant activation of Akt/mammalian target of rapamycin (mTOR) pathway is a frequent event in head and neck squamous cell carcinomas (HNSCC), thus representing a potential molecular target for the treatment of HNSCC patients. The ability to translate this emerging body of information into effective therapeutic strategies, however, has been hampered by the limited availability of animal models for oral malignancies. Here, we show that the administration in the drinking water to mice of 4-nitroquinoline-1 oxide, a DNA adduct-forming agent that serves as a surrogate of tobacco exposure, leads to the progressive appearance of preneoplastic and tumoral lesions in the tongue and oral mucosa, with 100% incidence after only 16 weeks of carcinogen exposure. Remarkably, many of these lesions evolve spontaneously into highly malignant SCCs few weeks after 4-nitroquinoline-1 oxide withdrawal. In this model, we have observed that the activation of the Akt-mTOR biochemical route represents an early event, which is already detectable in dysplastic lesions. Furthermore, we show that the inhibition of mTOR by the chronic administration of rapamycin halts the malignant conversion of precancerous lesions and promotes the regression of advanced carcinogen-induced SCCs. Together, these findings support the contribution of the mTOR signaling pathway to HNSCC progression and provide a strong rationale for the early evaluation of mTOR inhibitors as a molecular-targeted strategy for HNSCC chemoprevention and treatment.

Purpose: To recapitulate the generation of cancer stem cells in the context of an intact animal using a retroviral vector capable of in vivo delivery of oncogenes to primitive endothelial and hematopoietic stem cells., Experimental Design: Targeting of these progenitors was achieved using transgenic mice in which the avian TVA retroviral receptor was placed under the control of the stem cell leukemia (scl/tal-1) gene promoter and SCL +19 enhancer., Results: Injection of an avian retrovirus encoding polyoma middle T (PyMT), an oncogene that transforms endothelial cells, caused rapid lethality in all SCL-TVA mice but not in control TVA(-) littermates. The infected animals exhibited hemorrhagic foci in several organs. Histopathologic analysis confirmed the presence of hemangiomas and the endothelial origin of the PyMT-transformed cells. Surprisingly, the transformed endothelial cells contained readily detectable numbers of TVA(+) cells. By contrast, normal blood vessels had very few of these cells. The presence of TVA(+) cells in the lesions suggests that the cells originally infected by PyMT retained stem cell characteristics. Further analysis showed that the tumor cells exhibited activation of the phosphatidylinositol 3-kinase/Akt and S6/mammalian target of rapamycin pathways, suggesting a mechanism used by PyMT to transform endothelial progenitors in vivo., Conclusions: We conclude that this experimental system can specifically deliver oncogenes to vascular endothelial progenitors in vivo and cause a fatal neoplastic disease. This animal model should allow the generation of endothelial cancer stem cells in the natural environment of an immunocompetent animal, thereby enabling the recapitulation of genetic alterations that are responsible for the initiation and progression of human malignancies of endothelial origin.

Purpose: Squamous cell carcinoma of the head and neck (HNSCC), the sixth most prevalent cancer among men worldwide, is associated with poor prognosis, which has improved only marginally over the past three decades. A proteomic analysis of HNSCC lesions may help identify novel molecular targets for the early detection, prevention, and treatment of HNSCC., Experimental Design: Laser capture microdissection was combined with recently developed techniques for protein extraction from formalin-fixed paraffin-embedded (FFPE) tissues and a novel proteomics platform. Approximately 20,000 cells procured from FFPE tissue sections of normal oral epithelium and well, moderately, and poorly differentiated HNSCC were processed for mass spectrometry and bioinformatic analysis., Results: A large number of proteins expressed in normal oral epithelium and HNSCC, including cytokeratins, intermediate filaments, differentiation markers, and proteins involved in stem cell maintenance, signal transduction, migration, cell cycle regulation, growth and angiogenesis, matrix degradation, and proteins with tumor suppressive and oncogenic potential, were readily detected. Of interest, the relative expression of many of these molecules followed a distinct pattern in normal squamous epithelia and well, moderately, and poorly differentiated HNSCC tumor tissues. Representative proteins were further validated using immunohistochemical studies in HNSCC tissue sections and tissue microarrays., Conclusions: The ability to combine laser capture microdissection and in-depth proteomic analysis of FFPE tissues provided a wealth of information regarding the nature of the proteins expressed in normal squamous epithelium and during HNSCC progression, which may allow the development of novel biomarkers of diagnostic and prognostic value and the identification of novel targets for therapeutic intervention in HNSCC.

Purpose: As an approach to evaluate the expression pattern and status of activation of signaling pathways in clinical specimens from head and neck squamous cell carcinoma (HNSCC) patients, we established the Head and Neck Cancer Tissue Array Initiative, an international consortium aimed at developing a high-density HNSCC tissue microarray, with a high representation of oral squamous cell carcinoma., Experimental Design: These tissue arrays were constructed by acquiring cylindrical biopsies from multiple individual tumor tissues and transferring them into tissue microarray blocks. From a total of 1,300 cases, 547 cores, including controls, were selected and used to build the array., Results: Emerging information by the use of phosphospecific antibodies detecting the activated state of signaling molecules indicates that the Akt-mammalian target of rapamycin (mTOR) pathway is frequently activated in HNSCC, but independently from the activation of epidermal growth factor receptor or the detection of mutant p53. Indeed, we identified a large group of tissue samples displaying active Akt and mTOR in the absence of epidermal growth factor receptor activation. Furthermore, we have also identified a small subgroup of patients in which the mTOR pathway is activated but not Akt, suggesting the existence of an Akt-independent signaling route stimulating mTOR., Conclusions: These findings provide important information about the nature of the dysregulated signaling networks in HNSCC and may also provide the rationale for the future development of novel mechanism-based therapies for HNSCC patients.

Despite recent emerging evidence suggesting that cancer stem cells subsist in a variety of tumors, it is not yet fully elucidated whether postnatal stem cells are directly involved in tumorigenesis. We used murine bone marrow-derived mesenchymal stem cells (BMMSCs) as a model to test a hypothesis that tumorigenesis may originate from spontaneous mutation of stem cells. In this study, we demonstrated that murine BMMSCs, after numerous passages, obtained unlimited population doublings and proceeded to a malignant transformation state, resulting in fibrosarcoma formation in vivo. Transformed BMMSCs colonized to multiple organs when delivered systemically through the tail vein. Fibrosarcoma cells formed by transformed BMMSCs contained cancer progenitors, which were capable of generating colony clusters in vitro and fibrosarcoma in vivo by the second administration. The mechanism by which BMMSCs transformed to malignant cells was associated with accumulated chromosomal abnormalities, gradual elevation in telomerase activity, and increased c-myc expression. Moreover, BMMSCs and their transformed counterpart, fibrosarcoma-forming cells, demonstrated different sensitivity to anti-cancer drugs. BMMSCs/fibrosarcoma transformation system may provide an ideal system to elucidate the mechanism of how stem cells become cancer cells and to screen anti-sarcoma drugs.

Emerging knowledge on how the dysregulated function of signaling networks contributes to the malignant growth of squamous cell carcinoma of the head and neck (HNSCC) can now be exploited to identify novel mechanism-based anticancer treatments. In this regard, we have observed that persistent activation of the serine/threonine kinase Akt is a frequent event in HNSCC, and that blockade of its upstream kinase, 3'-phosphoinositide-dependent kinase 1, potently inhibits tumor cell growth. Akt promotes cell proliferation by its ability to coordinate mitogenic signaling with energy- and nutrient-sensing pathways that control protein synthesis through the atypical serine/threonine kinase, mammalian target of rapamycin (mTOR). This kinase, in turn, phosphorylates key eukaryotic translation regulators, including p70-S6 kinase and the eukaryotic translation initiation factor, 4E binding protein 1. Indeed, we show here that aberrant accumulation of the phosphorylated active form of S6, the most downstream target of the Akt-mTOR-p70-S6 kinase pathway, is a frequent event in clinical specimens from patients with HNSCC and their derived cell lines. Of interest, this enhanced level of the phosphorylated active form of S6 was rapidly reduced in HNSCC cell lines and HNSCC xenograft models at clinically relevant doses of rapamycin, which specifically inhibits mTOR. Furthermore, we observed that rapamycin displays a potent antitumor effect in vivo, as it inhibits DNA synthesis and induces the apoptotic death of HNSCC cells, ultimately resulting in tumor regression. These findings identify the Akt-mTOR pathway as a potential therapeutic target for HNSCC, and may provide the rationale for the early clinical evaluation of rapamycin and its analogues in patients with HNSCC.

We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/Endo180 is widely expressed in periductal fibroblast-like mesenchymal cells that line mammary epithelial cells. This pattern of uPARAP/Endo180 expression is preserved during polyomavirus middle T-induced mammary carcinogenesis, with strong uPARAP/Endo180 expression by mesenchymal cells embedded within the collagenous stroma surrounding nests of uPARAP/Endo180-negative tumor cells. Genetic ablation of uPARAP/Endo180 impaired collagen turnover that is critical to tumor expansion, as evidenced by the abrogation of cellular collagen uptake, tumor fibrosis, and blunted tumor growth. These studies identify uPARAP/Endo180 as a key mediator of collagen turnover in a pathophysiological context.

In this paper we demonstrate, using the C7-2-HI metastatic transplantable ductal mammary tumor, that endocrine therapy can induce complete regression of spontaneous lymph node and lung metastases in a mouse model of breast cancer progression. This tumor expresses high levels of estrogen and progesterone receptors and shows a high incidence of early axillary lymph nodes and lung metastases; using this model we had previously shown complete tumor regression of subcutaneous implants. Interestingly, although the metastases showed a more differentiated histology as compared with the primary growth, they underwent complete regression when treated with estrogens or antiprogestins. This phenomenon was associated with sustained cytostasis and apoptosis accompanied by increases in p21 and p27 expression and early tissue remodeling. These results highlight the essential role of PR in regulating cell proliferation in this model as well as its possible use as therapeutic target.

Interactions between steroid hormone receptors and signal transducer and activator of transcription (Stat)-mediated signaling pathways have already been described. In the present study, we explored the capacity of progestins to modulate Stat3 transcriptional activation in an experimental model of hormonal carcinogenesis in which the synthetic progestin medroxyprogesterone acetate (MPA) induced mammary adenocarcinomas in BALB/c mice and in the human breast cancer cell line T47D. We found that C4HD epithelial cells, from the MPA-induced mammary tumor model, expressed Stat3 and that MPA treatment of C4HD cells up-regulated Stat3 protein expression. In addition, MPA induced rapid, nongenomic Stat3, Jak1, and Jak2 tyrosine phosphorylation in C4HD and T47D cells. MPA treatment of C4HD cells also resulted in rapid c-Src tyrosine phosphorylation. These effects were completely abolished by the progestin antagonist RU486. Abrogation of Jak1 and Jak2 activity by transient transfection of C4HD cells with dominant negative (DN) Jak1 or DN Jak2 vectors, or inhibition of Src activity by preincubation of cells with the Src family kinase inhibitor PP2, blocked the capacity of MPA to induce Stat3 phosphorylation. Treatment of C4HD cells with MPA induced Stat3 binding to DNA. In addition, MPA promoted strong Stat3 transcriptional activation in C4HD and T47D cells that was inhibited by RU486 and by blockage of Jak1, Jak2, and Src activities. To investigate the correlation between MPA-induced Stat3 activation and cell growth, C4HD cells were transiently transfected with a DN Stat3 expression vector, Stat3Y705-F, or with a constitutively activated Stat3 mutant, Stat3-C. While expression of Stat3Y705-F mutant had an inhibitory effect on MPA-induced growth of C4HD cells, transfection with the constitutively activated Stat3-C vector resulted in MPA-independent proliferation. Finally, we addressed the effect of targeting Stat3 in in vivo growth of C4HD breast tumors. Blockage of Stat3 activation by transfection of C4HD cells with the DN Stat3Y705-F expression vector significantly inhibited these cells' ability to form tumors in syngeneic mice. Our results have for the first time demonstrated that progestins are able to induce Stat3 transcriptional activation, which is in turn an obligatory requirement for progestin stimulation of both in vitro and in vivo breast cancer growth.