Development and Light Response of Leaves of Metasequoia and Close Relatives

Metasequoia glyptostroboides is a useful nearest living relative (NLR) of the Eocene fossil Metasequoia. Research on modern Metasequoia might give us some clues about its fossil counterpart. During this study the leaf anatomy of Metasequoia, Glyptostrobus, Sequoia and Taxodium was investigated with light microscopy and transmission electron microscopy. Metasequoia exhibits several characteristics of typical sciaphilic plants, such as slightly arched outer cell walls in the adaxial epidermal cells, strongly arched outer cell walls in the abaxial epidermal cells, mesophyll composed of spongy cells, chloroplasts with well-developed grana not only in mesophyll cells but in both the adaxial and abaxial epidermis. Based on comparison of leaf morphology and anatomy, we conclude that Metasequoia is best adapted to low light intensities, Sequoia and Taxodium are intermediate, and Glyptostrobus is adapted to higher light intensities. The effects of light intensity on mesophyll plastids of Metasequoia leaves were studied with trees grown under different light intensities. Metasequoia had the ability to synthesize chlorophyll under complete darkness and was stressed under high light. These characteristics would provide adaptive advantages for Metasequoia to adapt to low intensity, low angle, polar light at their Eocene high latitude paleoenvironments, particularly during the polar spring when light levels are exceedingly low. It provides evidence to explain why Metasequoia was the dominant tree species in Eocene high latitudes. The thesis is written as an article to be submitted to the American Journal of Botany.

Emodin, a tyrosine kinase inhibitor, and human cancer

Many human diseases, including cancers, result from aberrations of signal transduction pathways. The recent understanding of the molecular biochemistry of signal transduction in normal and transformed cells enable us to have a better insight about cancer and design new drugs to target this abnormal signaling in the cancer cells. Tyrosine kinase pathway plays a very important role in normal and cancer cells. Enhanced activity of tyrosine kinases has been associated with many human cancer types. Therefore, identifying the type of tyrosine kinases involved in a particular cancer type and blocking these tyrosine kinase pathways may provide a way to treat cancer. Receptor tyrosine kinase expression, namely epidermal growth factor receptor (EGFR) family, was examined in the oral squamous cell carcinoma patients. The expression levels of different members of the EGFR family were found to be significantly associated with shorter patients' survival. Combining EGFR, HER-2/neu, and HER-3 expression can significantly improve the predicting power. The effect of emodin, a tyrosine kinase inhibitor, on these receptors in head and neck squamous cell carcinoma cell lines was examined. Emodin was found to suppress the tyrosine phosphorylation of HER-2/neu and EGF-induced tyrosine phosphorylation of EGFR. Emodin also induced apoptosis and downregulated the expression of anti-apoptotic protein bcl-2 in oral squamous cell carcinoma cells. It is known that tyrosine kinase pathways are involved in estrogen receptor signaling pathway. Therefore, the effects of inhibiting the tyrosine kinase pathway in estrogen receptor-positive breast cancers was studied. Emodin was found to act similarly to antiestrogens, capable of inhibiting estrogen-stimulated growth and DNA synthesis, and the phosphorylation of Rb protein. Interestingly, emodin, and other tyrosine kinase inhibitors, such as RG 13022 and genistein, depleted cellular levels of estrogen receptor protein. Emodin-induced depletion of estrogen receptor was mediated by the proteasome degradation pathway. In summary, we have demonstrated that tyrosine kinase pathways play an important role in oral squamous cell carcinoma and estrogen receptor-positive breast cancer. Targeting the tyrosine kinases by inhibitors, such as emodin, may provide a potential way to treat the cancer patients. ^

Role of the cyclin dependent kinase-4 in normal and neoplastic proliferation

In the last few years, our laboratory has studied the regulatory mechanisms of proliferation and differentiation in epidermal tissues. Our results showed differences in the roles of cyclin dependent-kinases 4 and 6, and the three D-type cyclins, during normal epidermal proliferation and neoplastic development. Thus, to elucidate the role of the different cell cycle regulators, we developed transgenic mice that overexpress CDK4 (K5-CDK4), or their cognate D-type cyclins, in epithelial tissues. The most severe phenotype was observed in K5-CDK4 animals that developed dermal fibrosis, epidermal hyperplasia and hypertrophy. Forced expression of CDK4 in the epidermal basal cell layer increased the malignant conversion of skin papillomas to squamous cell carcinomas (SCC). Contrastingly, lack of CDK4 completely inhibited tumor development, suggesting that CDK4 is required in this process. Biochemical studies demonstrated that p21 Cip1 and p27Kip1 inhibitors are sequestered by CDK4 resulting in indirect activation of Cyclin E/CDK2, implicating the non-catalytic activity of CDK4 in deregulation of the cell cycle progression. ^ It has been proposed that the proliferative and oncogenic role of Myc is linked to its ability to induce the transcription of CDK4, cyclin D1, and cyclin D2 in vitro. Deregulation of Myc oncogene has been found in several human cancers. Also it has been demonstrated that CDK4 has the ability to functionally inactivate the product of the tumor suppressor gene Rb, providing a link between Myc and the CDK4/cyclin D1/pRb/p16 pathway in some malignant tumors. Here, we sought to determine the role of CDK4 as a mediator of Myc activities by developing a Myc overexpressing mouse nullizygous for CDK4. We demonstrated that lack of CDK4 results in reduced keratinocyte proliferation and epidermal thickness in K5-Myc/CDK4-null mice. In addition, complete reversion of tumor development was observed. All together, this work demonstrates that CDK4 acts as an oncogene independent of the D-type cyclin levels and it is an important mediator of the tumorigenesis induced by Myc. In addition, we showed that the sequestering activity of CDK4 is critical for the development of epidermal hyperplasia during normal proliferation, malignant progression from papillomas to squamous cell carcinomas, and tumorigenesis induced by Myc. ^

Characterization of E2F1's oncogenic, apoptotic and tumor-suppressive activities utilizing the K5 mouse model

Though E2F1 is deregulated in most human cancers by mutations of the p16-cyclin D-Rb pathway, it also exhibits tumor suppressive activity. A transgenic mouse model overexpressing E2F1 under the control of the bovine keratin 5 (K5) promoter exhibits epidermal hyperplasia and spontaneously develops tumors in the skin and other epithelial tissues after one year of age. In a p53-deficient background, aberrant apoptosis in K5 E2F1 transgenic epidermis is reduced and tumorigenesis is accelerated. In sharp contrast, K5 E2F1 transgenic mice are resistant to papilloma formation in the DMBA/TPA two-stage carcinogenesis protocol. K5 E2F4 and K5 DP1 transgenic mice were also characterized and both display epidermal hyperplasia but do not develop spontaneous tumors even in cooperation with p53 deficiency. These transgenic mice do not have increased levels of apoptosis in their skin and are more susceptible to papilloma formation in the two-stage carcinogenesis model. These studies show that deregulated proliferation does not necessarily lead to tumor formation and that the ability to suppress skin carcinogenesis is unique to E2F1. E2F1 can also suppress skin carcinogenesis when okadaic acid is used as the tumor promoter and when a pre-initiated mouse model is used, demonstrating that E2F1's tumor suppressive activity is not specific for TPA and occurs at the promotion stage. E2F1 was thought to induce p53-dependent apoptosis through upregulation of p19ARF tumor suppressor, which inhibits mdm2-mediated p53 degradation. Consistent with in vitro studies, the overexpression of E2F1 in mouse skin results in the transcriptional activation of the p19ARF and the accumulation of p53. Inactivation of either p19ARF or p53 restores the sensitivity of K5 E2F1 transgenic mice to DMBA/TPA carcinogenesis, demonstrating that an intact p19ARF-p53 pathway is necessary for E2F1 to suppress carcinogenesis. Surprisingly, while p53 is required for E2F1 to induce apoptosis in mouse skin, p19ARF is not, and inactivation of p19ARF actually enhances E2F1-induced apoptosis and proliferation in transgenic epidermis. This indicates that ARF is important for E2F1-induced tumor suppression but not apoptosis. Senescence is another potential mechanism of tumor suppression that involves p53 and p19ARF. K5 E2F1 transgenic mice initiated with DMBA and treated with TPA show an increased number of senescence cells in their epidermis. These experiments demonstrate that E2F1's unique tumor suppressive activity in two-stage skin carcinogenesis can be genetically separated from E2F1-induced apoptosis and suggest that senescence utilizing the p19ARF-p53 pathway plays a role in tumor suppression by E2F1. ^

The role of Akt activation in mouse skin tumor promotion

The importance of IGF-1/IGF-1R signaling is evident in human cancers including breast, colon, prostate, and lung which have been shown to overexpress IGF-1. Also, serum levels of IGF-1 have been identified as a risk factor for these cancers. IGF-1 has been primarily shown to mediate its mitogenic effects through signaling pathways such as MAPK and PI3K/Akt. In this regard, BK5.IGF-1 transgenic mice were generated and these mice displayed hyperplasia and hyperkeratosis in the epidermis. In addition, these mice were also found to have elevated MAPK, PI3K, and Akt activities. Furthermore, overexpression of IGF-1 in epidermis can act as a tumor promoter. BK5.IGF-1 transgenic mice developed papillomas after initiation with DMBA without further treatment with a tumor promoter such as TPA. Previous data has also shown that inhibition of the PI3K/Akt signaling pathway by the inhibitor LY294002 was able to reduce the number of tumors formed by IGF-1 mediated tumor promotion. The current studies presented demonstrate that Akt may be the critical effector molecule in IGF-1/IGF-1R mediated tumor promotion. We have found that inhibition of PI3K/Akt by LY294002 inhibits cell cycle components, particularly those associated with G1 to S phase transition including Cyclin D1, Cyclin E, E2F1, and E2F4, that are elevated in epidermis of BK5.IGF-1 transgenic mice. We have also demonstrated that Akt activation may be a central theme in early tumor promotion. In this regard, treatment with diverse tumor promoters such as TPA, okadaic acid, chrysarobin, and UVB was shown to activate epidermal Akt and its downstream signaling pathways after a single treatment. Furthermore, overexpression of Akt targeted to the basal cells of the epidermis led to hyperplasia and increased labeling index as determined by BrdU staining. These mice also had constitutively elevated levels of cell cycle components, particularly Cyclin D1, Cyclin E, E2F1, E2F4, and Mdm-2. These mice developed skin tumors following initiation with DMBA and were hypersensitive to the tumor promoting effects of TPA. Collectively, these studies provide evidence that Akt activation plays an important role in the process of mouse skin tumor promotion. ^

The characterization of PKC-isozyme specific EGFR-dependent Erk1/2 activation in androgen-independent prostate cancer cell lines

One growth factor receptor commonly altered during prostate tumor progression is the epidermal growth factor receptor (EGFR). EGFR signaling regulates Erk1/2 phosphorylation through multiple mechanisms. We hypothesized that PKC isozymes play a role in EGFR-dependent signaling, and that through PKC isozyme selective inhibition, EGFR-dependent Erk1/2 activation can be attenuated in AICaP cells. ^ To test the hypothesis, PKC activation was induced by 12-O-tetradecanoyi-phorbol-13-acetate (TPA) in PC-3 cells. As a result, Erk1/2 was activated similarly to what was observed upon EGF stimulation. EGF-induced Erk1/2 activation in PC-3 cells was PKC-dependent, as demonstrated through use of a selective PKC inhibitor, GF109203X. This provides evidence for PKC regulatory control over Erk1/2 signaling downstream of EGFR. Next, we demonstrated that when PKC was inhibited by GF109203X, EGF-stimulated Erk1/2 activation was inhibited in PC-3, but not DU145 cells. TPA-stimulated Erk1/2 activation was EGFR-dependent in both DU145 and PC-3 cells, demonstrated through abrogation of Erk1/2 activation by a selective EGFR inhibitor AG1478. These data support PKC control at or upstream of EGFR in AICaP cells. We observed that interfering with ligand/EGFR binding abrogated Erk1/2 signaling in TPA-stimulated cells, revealing a role for PKC upstream of EGFR. ^ Next, we determined which PKC isozymes might be responsible for Erk1/2 regulation. We first determined that human AICaP cell lines express the same PKC isozymes as those observed in clinical prostate cancer specimens (α, ϵ, &zgr;, ι and PKD). Isozyme-selective methods were employed to characterize discrete PKC isozyme function in EGFR-dependent Erk1/2 activation. Pharmacologic inhibitors implicated PKCα in TPA-induced EGFR-dependent Erk1/2 activation in both PC-3 and DU145 cells. Further, the cPKC-specific inhibitor, Gö6976 decreased viablilty of DU145 cells, providing evidence that PKCα is necessary for growth and survival. Finally, resveratrol, a phytochemical with strong cancer therapeutic potential inhibited Erk1/2 activation, and this correlated with selective inhibition of PKCα. These results demonstrate that PKC regulates pathways critical to progression of CaP cells, including those mediated by EGFR. Thus, PKC isozyme-selective targeting is an attractive therapeutic strategy, and understanding the role of specific PKC isozymes in CaP cell growth and survival may aid in development of effective, non-toxic PKC-targeted therapies. ^

Tumor heterogeneity dictates sensitivity to gefitinib (Iressa(TM)/ZD1839) in solid tumor models

The epidermal growth factor receptor (EGFR) and its ligands are overexpressed in many human tumors, including bladder and pancreas, correlating with a more aggressive tumor phenotype and poor patient prognosis. We initiated the present study to characterize the heterogeneity of gefitinib responsiveness in a panel of human bladder and pancreatic cancer cell lines in order to identify the biological characteristics of EGFR-dependent proliferation that could be used to prospectively identify drug-sensitive tumors. A second objective was to elucidate how to best exploit these results by utilizing gefitinib in combination therapy. To these ends, we examined the effects of the EGFR antagonist gefitinib on proliferation and apoptosis in a panel of 18 human bladder cancer cell lines and 9 human pancreatic cancer cell lines. Our data confirmed the existence of marked heterogeneity in Iressa responsiveness with less than half of the cell lines displaying significant growth inhibition by clinically relevant concentrations of the drug. Gefitinib responsiveness was found to be p27 kip1 dependent as DNA synthesis was restored following exposure to p27siRNA. Unfortunately, Iressa responsiveness was not closely linked to surface EGFR or TGF-α expression in the bladder cancer cells, however, cellular TGF-α expression correlated directly with Iressa sensitivity in the pancreatic cancer cell lines. These findings provide the potential for prospectively identifying patients with drug-sensitive tumors. ^ Further studies aimed at exploiting gefitinib-mediated cell cycle effects led us to investigate if gefitinib-mediated TRAIL sensitization correlated with increased p27kip1 accumulation. We observed that increased TRAIL sensitivity following gefitinib exposure was not dependent on p27 kip1 expression. Additional studies initiated to examine the role(s) of Akt and Erk signaling demonstrated that exposure to PI3K or MEK inhibitors significantly enhanced TRAIL-induced apoptosis at concentrations that block target phosphorylation. Furthermore, combinations of TRAIL and the PI3K or MEK inhibitors increased procaspase-8 processing above levels observed with TRAIL alone, indicating that the effects were exerted at the level of caspase-8 activation, considered the earliest step in the TRAIL pathway. ^

Significance of signal transducer and activator of transcription 3 during multistage mouse skin carcinogenesis

Signal transducer and activator of transcription 3 (Stat3) is a signaling molecule that transduces signal from cell surface receptors, itself translocates into the nucleus, binds to consensus promoter sequences and activates gene transcription. Here, we showed that Stat3 is constitutively activated in both premalignant tumors (papillomas) and squamous cell carcinomas of mouse skin that is induced by topical treatment with an initiator 7,12-dimethylbenz[a]anthracene (DMBA) followed by a tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Additional data demonstrated that epidermal growth factor signaling contributes to the activation of Stat3 in this model. Using mice where Stat3 function is abrogated in keratinocytes via the Cre-LoxP system (K5Cre.Stat3 flox/flox), we demonstrated that Stat3 is required for de novo carcinogenesis since Stat3 deficiency leads to a complete abrogation of skin tumor development induced by DMBA and TPA. We subsequently showed that Stat3 plays a role in both the initiation and promotion stages of carcinogenesis. During initiation, Stat3 functions as an anti-apoptotic molecule for maintaining the survival of DNA-damaged keratinocyte stem cells. During promotion, Stat3 functions as a critical regulator for G1 to S phase cell cycle progression to confer selective clonal expansion of initiated cells into papillomas. On the other hand, using transgenic mice over-expressing a constitutively dimerized form of Stat3 (Stat3C) in keratinocytes (K5.Stat3C), we revealed a role for Stat3 in tumor progression. After treatment with DMBA and TPA, K5.Stat3C transgenic mice developed skin tumors with a shorter latency when 100% bypassed the premalignant stage and became carcinoma in situ. Histological and immunohistochemical analysis revealed these tumors as highly vascularized and poorly differentiated. More strikingly, these tumors exhibited invasion into surrounding mesenchymal tissue, some of which metastasized into lung. The tumor-mesenchymal front was characterized by partial loss of E-cadherin and elevation of vimentin, markers characterizing epithelial-mesenchymal transition. On the other hand, inhibition of Stat3 via a decoy oligonucleotide led to a significant reduction of tumor size in approximately 50% of all papillomas tested. In conclusion, we demonstrated that Stat3 plays a critical in all three stages (initiation, promotion and progression) of skin carcinogenesis, and it may potentially become a good target for cancer prevention and anti-cancer therapy. ^

Homeostatic transcriptional regulation of bcl-2 in the epidermis

Bcl-2, a crucial regulator of cell survival, is frequently overexpressed in basal cell carcinomas (BCCs), the most commonly diagnosed cancers. Regulation of bcl-2 expression in epidermal keratinocytes is not well characterized. In the epidermis, bcl-2 is expressed only in keratinocytes of the basal layer and the outer root sheath of hair follicles and no bcl-2 expression in suprabasalar keratinocytes. The calcium gradient in the epidermis is a potent regulator of keratinocyte differentiation. Increasing calcium concentrations associated with differentiation, resulted in the downregulation of a 2.9 kb bcl-2 promoter luciferase construct. The AP-1 family of transcription factors is differentially expressed in the strata of the epidermis and has been shown to be involved in the stage specific expression of numerous differentiation markers in the epidermis. In silico analysis of the bcl-2 promoter and gene reporter assays showed that co-transfection of JUNB and JUND, but not other AP-1 dimers, caused a significant upregulation of the bcl-2 promoter in primary keratinocytes. Immunoelectrophoretic mobility shift assays, in vivo chromatin immunoprecipitation (ChIP) studies and mutational analysis of AP-1 binding site 3 on the bcl-2 promoter identified it as the site involved in bcl-2 regulation. Utilizing site directed mutants, we determined that phosphorylation at Ser90/Ser100 residues of JUND is required for the activation of the bcl-2 promoter. ^ The sonic hedgehog (SHH) pathway is frequently deregulated in BCCs and, we have shown that GLI1 upregulates bcl-2 in keratinocytes. While examining potential regulation of the SHH pathway extracellular calcium, we found that higher calcium concentrations are associated with lowered HH pathway activity and upregulation of suppressor of fused (SUFU) which negatively regulates the SHH pathway. ChIP assays, and in vivo mouse models, show that ΔNp63α, a crucial regulator of epidermal development, binds and activates the SUFU promoter in differentiating keratinocytes. Increasing SUFU levels prevent transactivation of the bcl-2 promoter. In vitro SUFU knockdown along with in vivo SUFU+/− murine models demonstrate a significant upregulation of bcl-2 expression. ^ In conclusion, the spatial and temporal expression of bcl-2 during keratinocyte differentiation in the epidermis is a complex process requiring cooperative interactions of specific signaling cascades and transcription factors. ^

Molecular mechanism of jet fuel induced immune suppression

Dermal exposure to jet fuel suppresses the immune response. Immune regulatory cytokines, and biological modifiers, including platelet activating factor, prostaglandin E2, and interleukin-10 have all been implicated in the pathway leading to immunosuppression. It is estimated that approximately 260 different hydrocarbons are found in JP-8 (jet propulsion-8) jet fuel, and the identity of the immunotoxic compound is not known. The recent availability of synthetic jet fuel (S-8), which is devoid of aromatic hydrocarbons, made it feasible to design experiments to test the hypothesis that the aromatic hydrocarbons are responsible for jet fuel induced immune suppression. Applying S-8 to the skin of mice does not up-regulate the expression of epidermal cyclooxygenase-2 nor does it induce immune suppression. Adding back a cocktail of 7 of the most prevalent aromatic hydrocarbons found in jet fuel to S-8 up-regulated cyclooxygenase-2 expression and induced immune suppression. Cyclooxygenase-2 induction can be initiated by reactive oxygen species (ROS). JP-8 treated keratinocytes increased ROS production, S-8 did not. Antioxidant pre-treatment blocked jet fuel induced immune suppression and cyclooxygenase-2 up-regulation. Accumulation of reactive oxygen species induces oxidant stress and affects activity of ROS sensitive transcription factors. JP-8 induced activation of NFκB while S-8 did not. Pre-treatment with antioxidants blocked activation of NFκB and parthenolide, an NFκB inhibitor, blocked jet fuel induced immune suppression and cyclooxygenase-2 expression in skin of treated mice. p65 siRNA transfected keratinocytes demonstrated NFκB is critically involved in jet fuel induced COX-2 expression. These findings clearly implicate the aromatic hydrocarbons found in jet fuel as the agents responsible for inducing immune suppression, in part by the production of reaction oxygen species, NFκB dependent up-regulation of cyclooxygenase-2, and the production of immune regulatory factors and cytokines. ^

Near infra-red-activatable nanocarriers for selective cancer diagnosis and treatment

Standard treatment strategies for cancer patients include surgery, radiation therapy, and chemotherapy. Although these strategies have been proven effective, they also have associated limitations. An attractive and innovative approach that can be used alone or in combination with the above modalities is based on the systemic or topical administration of a nanomaterial-based photoactive compound. Interaction with light in the near infrared (NIR) region results in either emission of fluorescence, which can be used for photodetection, or absorption of light which results in phototherapy. Nanomaterials have the advantage of providing multi-functional and unique properties in a single device that cannot be readily acquired with conventional small molecular weight compounds. ^ In this study, three different novel nanocarrier systems were designed and evaluated in mediating photodetection and phototherapy in the NIR. The first compound synthesized was a dual-labeled magnetic resonance/optical imaging agent for sentinel lymph node mapping and biopsy. This dual-labeled agent combines the high resolution of magnetic resonance imaging with the highly sensitive detection of optical imaging. The second imaging agent was an activatable optical imaging agent used to monitor cathepsin B activity in vivo and to probe the degradation of poly(L-glutamic acid). This polymeric nanocarrier offers highly sensitive technique for the detection of enzymatic activity, with is not yet possible with small molecular weight compounds. The third agent was a C225-conjugated hollow nanoshell that is targeted to epidermal growth factor receptors. This targeting agent has been demonstrated to mediate photothermal therapy both in vitro and in vivo. ^ These nanocarrier systems are an invaluable tool for the detection of cancer and many other diseases. With improved targeted delivery of these agents, the ability to diagnose diseases will become more sensitive and more specific. Finally, when designed properly, these agents would allow concurrent diagnosis and treatment of patients of various diseases. ^

Oncogenic activation of c-Abl tyrosine kinase in non-small cell lung cancer: FUS1 tumor suppressor down-regulates c-Abl tyrosine kinase

The FUS1 tumor suppressor gene (TSG) has been found to be deficient in many human non-small cell lung cancer (NSCLC) tissue samples and cell lines (1,2,3). Studies have shown potent anti-tumor activity of FUS1 in animal models where FUS1 was delivered through a liposomal vector (4) and the use of FUS1 as a therapeutic agent is currently being studied in clinical human trials (5). Currently, the mechanisms of FUS1 activity are being investigated and my studies have shown that c-Abl tyrosine kinase is inhibited by the FUS1 TSG.^ Considering that many NSCLC cell lines are FUS1 deficient, my studies further identified that FUS1 deficient NSCLC cells have an activated c-Abl tyrosine kinase. C-Abl is a known proto-oncogene and while c-Abl kinase is tightly regulated in normal cells, constitutively active Abl kinase is known to contribute to the oncogenic phenotype in some types of hematopoietic cancers. My studies show that the active c-Abl kinase contributes to the oncogenicity of NSCLC cells, particularly in tumors that are deficient in FUS1, and that c-Abl may prove to be a viable target in NSCLC therapy.^ Current studies have shown that growth factor receptors play a role in NSCLC. Over-expression of the epidermal growth factor receptor (EGFR) plays a significant role in aggressiveness of NSCLC. Current late stage treatments include EFGR tyrosine kinase inhibitors or EGFR antibodies. Platelet-derived growth factor receptor (PDGFR) also has been shown to play a role in NSCLC. Of note, both growth factor receptors are known upstream activators of c-Abl kinase. My studies indicate that growth factor receptor simulation along deficiency in FUS1 expression contributes to the activation of c-Abl kinase in NSCLC cells. ^

The transcriptional repressor Delta EF1 controls resistance to the EGFR inhibitor erlotinib in human HNSCC lines

Epidermal Growth Factor Receptor (EGFR) overexpression occurs in about 90% of Head and Neck Squamous Cell Carcinoma (HNSCC) cases. Aberrant EGFR signaling has been implicated in the malignant features of HNSCC. Thus, EGFR appears to be a logical therapeutic target with increased tumor specificity for the treatment of HNSCC. Erlotinib, a small molecule tyrosine kinase inhibitor, specifically inhibits aberrant EGFR signaling in HNSCC. Only a minority of HNSCC patients were able to derive a substantial clinical benefit from erlotinib. ^ This dissertation identifies Epithelial to Mesenchymal Transition (EMT) as the biological marker that distinguishes EGFR-dependent (erlotinib-sensitive) tumors from the EGFR-independent (erlotinib-resistant) tumors. This will allow us to prospectively identify the patients who are most likely to benefit from EGFR-directed therapy. More importantly, our data identifies the transcriptional repressor DeltaEF1 as the mesenchymal marker that controls EMT phenotype and resistance to erlotinib in human HNSCC lines. si-RNA mediated knockdown of DeltaEF1 in the erlotinib-resistant lines resulted in reversal of the mesenchymal phenotype to an epithelial phenotype and significant increase in sensitivity to erlotinib. ^ DeltaEF1 represses the expression of the epithelial markers by recruiting HDACs to chromatin. This observation allows us to translate our findings into clinical application. To test whether the transcriptional repression by DeltaEF1 underlines the mechanism responsible for erlotinib resistance, erlotinib-resistant lines were treated with an HDAC inhibitor (SAHA) followed by erlotinib. This resulted in a synergistic effect and substantial increase in sensitivity to erlotinib in the resistant cell lines. Thus, combining an HDAC inhibitor with erlotinib represents a novel promising pharmacologic strategy for reversing resistance to erlotinib in HNSCC patients. ^

Role of activation of the protein tyrosine kinase, Src, in colorectal cancer chemoresistance

Advances in therapy for colorectal cancer have been hampered by development of resistance to chemotherapy. The Src family of protein tyrosine kinases has been associated with colorectal cancer development and progression. Activation of the prototypic member of the family, Src, occurs in advanced colorectal cancer and is associated with a worse outcome. This work tests the hypotheses that Src activation contributes to chemoresistance in some colon tumors and that this resistance can be overcome by use of Src inhibitors. The aims of the proposal were to (1) determine if constitutive Src activation is sufficient to induce oxaliplatin resistance; (2) evaluate the role of reactive oxygen species (ROS) in the activation of Src after oxaliplatin treatment; (3) determine the frequency of Src activation in liver metastases after oxaliplatin treatment; and (4) evaluate the safety, preliminary efficacy, and pharmacodynamics of the combination of dasatinib with oxaliplatin-based therapy in patients with metastatic colorectal cancer. ^ Using a panel of colon cancer cell lines and murine models, I demonstrate that administration of oxaliplatin, a commonly utilized chemotherapy for colorectal cancer, results in an increased activation of Src. The activation occurs acutely in some, but not all, colorectal carcinoma cell lines. Cell lines selected for oxaliplatin resistance are further increased in Src activity. Treatment of cell lines with dasatinib, a non-selective pharmacologic inhibitor of the Src family kinases synergistically killed some, but not all cell lines. Cell lines with the highest acute activation of Src after oxaliplatin administration were the most sensitive to the combination therapy. Previous work demonstrated that siRNA to Src increased sensitivity to oxaliplatin, suggesting that the effects of dasatinib are primarily due to its ability to inhibit Src in these cell lines. ^ To examine the mechanism underlying these results, I examined the effects of reactive oxygen species (ROS), as previous studies have demonstrated that platinum chemotherapeutics result in intracellular oxidative stress. I demonstrated that oxaliplatin-induced reactive oxygen species were higher in the cell lines with Src activation, relative to those in which Src was not activated. This oxaliplatin-induced Src activation was blocked by the administration of anti-oxidants, thereby demonstrating that synergistic killing between dasatinib and oxaliplatin was associated with the ability of the latter to generate ROS. ^ In a murine model of colorectal cancer metastasis to the liver, the combination of dasatinib and oxaliplatin was more effective in reducing tumor volume than either agent alone. However, when oxaliplatin resistant cell lines were treated with a combination of oxaliplatin and AZD0530, an inhibitor in the clinic with increased specificity for Src, no additional benefit was seen, although Src was activated by oxaliplatin and Src substrates were inhibited. The indolent growth of oxaliplatin-resistant cells, unlike the growth of oxaliplatin resistant tumors in patients, precludes definitive interpretation of these results. ^ To further explore Src activation in patients with oxaliplatin exposure and resistance, an immunohistochemistry analysis of tumor tissue from resected liver metastases of colorectal cancer was performed. Utilizing a tissue microarray, staining for phosphorylated Src and FAK demonstrated strong staining of tumor relative to stromal and normal liver. In patients recently exposed to oxaliplatin, there was increased FAK activation, supporting the clinical relevance of the prior preclinical studies. ^ To pursue the potential clinical benefit of the combination of Src inhibition with oxaliplatin, a phase IB clinical trial was completed. Thirty patients with refractory metastatic colorectal cancer were treated with a combination of 5-FU, oxaliplatin, an epidermal-growth factor receptor monoclonal antibody, and dasatinib. The recommended phase II dose of dasatinib was established, and toxicities were quantified. Pharmacodynamic studies demonstrated increased phosphorylation of the Src substrate paxillin after dasatinib therapy. Tumor biopsies were obtained and Src expression levels were quantitated. Clinical benefit was seen with the combination, including a response rate of 20% and disease control rate of 56%, prompting a larger clinical study. ^ In summary, although Src is constitutively activated in metastatic colorectal cancer, administration of oxaliplatin chemotherapy can further increase its activity, through a reactive oxygen species dependent manner. Inhibition of Src in combination with oxaliplatin provides additional benefit in vitro, in preclinical animal models, and in the clinic. Further study of Src inhibition in the clinic and identification of predictive biomarkers of response will be required to further advance this promising therapeutic target. ^

The role of chronic irritation and inflammation in tumor promotion in CD-1 mice by petroleum middle distillates

An initiation-promotion bioassay in CD-1 mice was used to examine the role of chronic irritation and inflammation in tumor promotion by petroleum middle distillates. A representative hydrodesulfurized middle distillate (API 81-07) was selected as the test article. Test groups (54 mice per group) were initiated once with 50 ug of 7,12-dimethylbenzanthracene (DMBA). Promotion with API 81-07 consisted of twice weekly treatments for 25 weeks with either 25 ul, 50 ul, 50 ul + daily treatment with 15 ug dexamethasone, 50 ul + post-application washings, and 100 ul. Three mice from each group were sacrificed at 21 day intervals (24 total per group). The skin from interim sacrificed (IS) mice was examined histopathologically for tumor, acanthosis, hyperkeratosis, pseudo-epitheliomatous hyperplasia, epidermal crusting, and subacute inflammation. In-life observations included examination of all mice for erythema and edema for 8 weeks following the first promotion treatment. Tumor incident at study termination was as follows: 25 ul (45%), 50 ul (43%), 50 ul + dexamethasone (0%), 50 ul + washing (70%), and 100 ul (81%). An overall correlation of $>$0.90 between tumor incidence and group means for acanthosis and hyperkeratosis was observed in IS mice at all intervals. Correlations of $<$0.75 were observed for other group mean histopathological parameters and tumor incidence. The overall correlation of group mean erythema and edema with tumor incident was $>$0.90. The results of this study support the hypothesis that induction of a lasting, albeit mild, hyperplasia is an essential, but not sufficient requirement, for tumor promotion. Furthermore, subacute inflammation does not appear to be a significant factor in tumor promotion by petroleum middle distillates. However, inflammation may be a factor in tumor progression. ^