A Novel Therapeutic Approach to Acral Lentiginous Melanoma Karen Melendez, Jorge Vargas, Silas Chan, Muska Hassan, Alex

Wesselhoeft

A Brief History: Karen Melendez

Acral lentignous melanoma (ALM) was first reported in 1976 by Reed (Park and Cho, 200) and is one of 4 subtypes of melanomas that develop from radically modified behavior of melanocyte cells. It accounts for approximately 1-3% of all melanoma cases and is primarily diagnosed in Asian, Black and Hispanic populations on non-hair bearing skin such as the palms, soles, finger and toe nails. Due to its rarity, ALM can be difficult to diagnose in early stages. Although it is a cutaneous melanoma, it is not associated with abnormal molar growth. Rather, ALM typically exhibits discoloration in nail beds and on the soles of feet, increasing the odds of misdiagnosis and affecting good prognosis (Park and Cho, 2010). Historically, ALM has been considered the most aggressive form of melanoma, however studies linked the lack of awareness to late diagnosis in situ, leading to its poor prognosis (Park and Cho, 2010). ALM, like other melanomas, needs to be confirmed and treated through a combination of approaches, including biopsy, histopathology, immunohistochemistry and dermascopy. Excision and amputation are the most common forms of treatment, however, local recurrence can occur, particularly in excisional biopsies and nail extraction methods. Amputation is an extreme approach, thus developing a method to attack the tumor locally without altering the external physiology of a patient and preventing the tumor from metastasizing is ideal.

Unlike Superficial spreading melanoma (SSM), Nodular melanoma (NM) and Lentigomaligna melanoma (LMM), ALM is not associated with sun exposure and damage; however, all melanomas exhibit altered levels of melanogenesis. In SSM, NM and LMM, melanogenesis rates are much higher in Caucasians than normal, whereas melanogensis in ALM patients is lower. Based on data presented by Porcia T. Bradford et al. in their 2009 article, Acral Lentiginous Melanoma: Incidence and Survival Patterns in the United States, 1986-2005, data suggests inhibited melanogenesis is linked to ethnology. This presents a challenge in developing overall melanoma treatments if ethnology is related to decreased melanogenesis in ALM because genes can have different mutation rates depending on the patients race. In Cancer Genes in Lung Cancer: Racial Disparities: Are There Any by Ahmed El-Telbany and Patrick Ma, they point out that these genomic landscape differences in the context of racial disparities should be emphasized both in tumorigenesis and in drug sensitivity/resistance. Darkly pigmented skin is a result of skin pigmentation genes and the production of eumelanin which is responsible for UV attenuation in darker pigmented individuals. Eumelanin production is induced by MITF, one of several genes that have high mutation rates in melanoma patients. In addition to MITF, CDKN2A, CDK4, MC1R, EGFR, MET, ERBB4, various Eph receptors, GRM3, GRIN2A , RAS, GNAQ, BRAF, KIT and MEK are important in melanocyte development and high frequency of mutations in these genes has been noted in melanomas. Loss of MITF function has detrimental effects on melanin production and the number of melanocytes (Uong and Zon, 2010) and frequently is overexpressed in melanomas (Garraway et al., 2005). MITF is upregulated and induced by increases from CREB in cAMPs response to MC1R binding to -MSH (Garcia-Borron et al., 2005). cKIT is a type III receptor tyrosine kinase

associated with pigmentation, and mutations in KIT affect the development, migration and survival of meloncytes (Uong and Zon, 2010). 36% of ALMs have amplified or mutated cKIT loci (Tsao et al., 2012). Snail/Slug, Sox 10 and endothelins are all important factors in early development of melanocytes from the neural crest that are responsible for protein binding. Lack of certain endothelian proteins, such as endothelin B-receptor can result in melanocytes not developing and mutations in protein receptors and their corresponding ligands are also common in melanomas (Uong and Zon, 2010). With an overwhelming possibility of pathways and genes to target, it is essential to take a deeper look at the genetic pathways involved to design a treatment plan.

Genetic Systems: Jorge Vargas

Acral lentiginous melanoma does not discriminate, as it is able to mutate many genes to bring about its malignancy. These mutations, however, almost always occur at some point in the cKIT/CREB signaling pathway. The cKIT/CREB pathway plays a pivotal role in cell growth and proliferation. Activation of this pathway results in gene activation involved with the accumulation of CDK4/Cyclin D1 complex (Swick et al., 2012). When this complex is present, the cell can progress through stages of proliferation. Previous attempts at an effective treatment have been insufficient in part because current drugs can only inhibit one protein at a time, and cancer cells can easily develop resistance by becoming too variable in the mutations they accrue. Tumor cells often mutate other elements of the cKIT/CREB pathway to overcome the drugs inhibiting them. For this reason, multiple proteins in this pathway have been targets for research and experimentation, and eventually for therapy. Such research to identify these proteins was carried out by the Comprehensive Cancer Center, which collected 102 samples of primary melanomas, twenty-eight of those samples being acral skin with ALM, and evaluated them for mutations or increased copy number of the cKIT receptor. After undergoing PCR and immunohistochemistry, samples with affected cKIT pathways were identified. Aberrations of cKIT were found in 36% of acral samples. The results brought more attention to cKIT as it was confirmed as an oncogene for ALM. Interestingly, mutations in BRAF were also found in 21% of samples. These melanoma types only infrequently show mutations in BRAF (Curtin et al., 2006). Because of these results, however, BRAF easily would stay a suspect to the origin of abnormalities.

In 2006, twenty-one samples of ALM were studied to calculate frequencies of BRAF and NRAS mutations. These mutations were identified through RFLP-PCR. BRAF mutations at exon 15 were found in 9.5% of samples. This was no surprise, seeing that abnormalities in BRAF were expected to be low. Mutations in NRAS at exon 2 were found in 42.9% of samples. Both of these genes are part of the cKIT pathway (Saldanha et al., 2006). In Spain seventeen samples of skin with ALM were collected from Spanish patients. The purpose of the experiment was to search the genomes in the affected DNA via PCR and identify the oncogene. The results shed light on which genes are mutated in the cancer, showing that AURKA, TERT, and NRAS genes are susceptible to mutations. The percentages of the samples with these mutations were 37.5, 31.2, and 25, respectively. There were no mutations at BRAF (Puig-Butille et al., 2013). Although, BRAF mutations are highly prevalent (59%) in melanomas occurring on skin without signs of chronic sun-induced damage (non-CSD melanomas), BRAF mutations occur significantly less frequent in melanomas on sun-protected skin such as the palms, soles, or subungual sites (acral melanomas), and on mucosal membranes (mucosal melanomas) (Curtin et al., 2006). The results have given this idea supports. BRAF, however, should not be written off as unrelated and insignificant. Previous experiments have proven that BRAF is as much of a target as the other proteins involved. Essentially, almost all of the proteins under scrutiny for targeted therapy have the common characteristic of being involved in the cKIT/CREB pathway. Because of the research that has been carried out, it is known that ALM targets the cKIT pathway. BRAF and NRAS have been reported as proteins susceptible to mutation, which further indicates that treatment with a single target may not be enough. One would need a treatment that would inhibit the entire

pathway. If this were accomplished, upstream mutations could be controlled for, resulting in a more effective therapy less prone to resistance.

Current Treatments: Muska Hassan

There are multiple therapies that a patient is given when diagnosed with cancer, such as chemotherapy, angiogenesis inhibitor and biochemotherapy, and palliative local therapy. Chemotherapy ideally slows or stops the growth of cancer cells. Biochemotherapy uses immunotherapy in conjunction with chemotherapy to shrink tumors more effectively than single agent or combination therapy. However, there is little evidence that this therapy is more effective at extending overall survival than single agent chemotherapy, combination therapy, or single agent immunotherapy (Ives et al., 2007). Melanomas that have not escaped the upper layer of the skin can be surgically removed. However, surgery comes along with some restrictions. The primary lesion can only be surgically removed if it is 2 mm or less in thickness (Veronesi and Cascinelli, 1991). A patient whose melanoma is more than 2 mm but less than 4 mm in thickness is considered for sentinel lymph node biopsy followed by complete lymph node dissection if the sentinel lymph nodes are microscopically or macroscopically positive (National Cancer Institute, 2012). Patients with a lesion larger than 4 mm in thickness are considered for adjuvant therapy. In this therapy, patients are given additional treatment after the primary treatment to lower their risk of being diagnosed with cancer again. These treatments may include chemotherapy, radiation therapy, hormone therapy, targeted therapy, or biological therapy (National Cancer Institute). Treating ALM with surgery can be very challenging due to functional requirements of these body parts. Therefore, alternative to surgery is amputation because of the poor prognosis due to a delay in diagnosis of this disease. This is because not many people are aware of this cancer type when it first occurs

(Franke et al., 2000; Bennett et al, 1994). Despite this radical surgical approach, cancer recurrence has been recorded in some cases. Although advances have been made in cancer therapies, the majority of patients are not cured. This is mainly because ALM often harbors multiple mutations in the cKIT/CREB pathway such as cKIT, BRAF/MEK, P13K, AKT, CDK4, NRAS and Cyclin D1. To inhibit these mutated proteins from continuously signaling the cell to proliferate, signal transduction inhibitors have been designed. These drugs include: Vemurafenib: BRAF inhibitor for patients with BRAFV600E melanomas (Sosman et al., 2012). BRAF is a member of the RAF kinase family and it regulates the MAP kinase/ERKs signaling pathway. This pathway leads to cell division, differentiation, and secretion. Due to the complexity of this pathway, vemurafenib is discouraged in some mutations because it may reduce BRAF activity in MAPK pathway but may adapt different characterization that may activate CRAF, which then signals ERK for reactivation of the MAPK pathway (Heidorn et al., 2010). Sorafenib: Multi-kinase inhibitor works against both the vascular endothelial growth factor signaling and the RAF/MEK pathway at the level of RAF kinase. This drug has not been successful because it has shown minimal activity in being a RAF kinase inhibitor and no improvement over chemotherapy alone as either first line treatment or second line treatment (Hauschild et al., 2009). CHIR-265 is a potential selective inhibitor of CRAF and mutant BRAF that inhibits cell proliferation and survival of cancer cell lines with NRAS/RAF pathway mutations. This drug is currently in phase I clinical trials (Amiri et al, 2006).

NVP-BEZ235 is an imidazo[4,5-c]quinoline derivative that inhibits PI3K and mTOR kinase activity by binding to the ATP-binding cleft of these enzymes. This drug is currently in phase I clinical trials (Maira et al., 2008). PD0325901 is an orally bioavailable, synthetic organic molecule targeting mitogenactivated protein kinase (MAPK/ERK kinase or MEK) with potential antineoplastic activity. This drug is currently in phase II clinical trials (Henderson et al., 2010). PD0332991 is an orally active, highly selective inhibitor of the cyclin D kinases CDK4 and CDK6 with ability to block retinoblastoma (Rb) phosphorylation in the low nanomolar range. This drug is currently in phase I clinical trials (Pfizer Clinical Trials, 2013). PI3K pathway is frequently mutated in human tumors, promoting cell proliferation, survival, and resistance to chemotherapy and radiotherapy. There are several PI3K inhibitors that are still in clinical trials and they include:

A. SF1126 is a phosphoinositide-3-kinase (PI3K) inhibitor and mammalian target of rapamycin (mTOR). This drug is currently in clinical I trial (Pfizer Clinical Trials, 2013)

B. BKM120 specifically inhibits class I PI3K in the P13K/AKT kinase-signaling pathway in an ATP-competitive manner, thereby inhibiting activation of the PI3K signaling pathway (Pfizer Clinical Trials, 2013)

C. CAL-101 is an orally bioavailable, small molecule inhibitor of the delta isoform of the PI3K with potential immunomodulating and antineoplastic activities. This drug is currently in phase II clinical trials (Pfizer Clinical Trials, 2013).

D. SAR245408 (XL147) is an orally available inhibitor of PI3K pathway. This drug is currently in phase I clinical trials (Pfizer Clinical Trials, 2013).

E. PI3K inhibitor PX-866 inhibits the production of the secondary messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3) and activation of the PI3K/Akt signaling pathway. This drug is currently in phase I clinical trials (Pfizer Clinical Trials, 2013).

A New Treatment; Proposed Target: Alex Wesselhoeft

The proposed target for an alternate therapy to be used in the treatment of ALM is the transcription factor CREB. Also known as cAMP response element-binding protein, CREB binds to DNA sequences to modulate the transcription of a number of genes including c-fos, neurotrophin BDNF, tyrosine hydroxylase and a number of neuropeptides. Transcription of these genes increases CDK4/Cyclin D1 complex formation. (Impey et al., 2004). As previously discussed, a large percentage of melanomas occur due to a number of possible mutations in the CREB pathway. The most common of these mutations occur in cKIT, BRAF, NRAS, and several others, all of which are intermediate steps of the CREB pathway (Swick et al., 2012). These mutations are often activating, drastically increasing downstream CREB activity and expression (Xiao et al., 2010). Increased CREB activity leads to increased and uncontrolled cell proliferation, one of the hallmarks of cancer, via excess CDK4/Cyclin D1 complex activity (Swick et al., 2012). While small molecule inhibitors of the listed mutated factors have achieved some clinical success, drug resistance, especially in BRAF mutant melanomas, is a common and inherent flaw in most, if not all, modern ALM chemotherapies (Thakur et al., 2013). To bypass the problem of drug resistance, we propose to inhibit CREB, the downstream target of melanomas most commonly mutated signaling pathway. A molecule that inhibits the eventual target of commonly mutated proteins like cKIT, BRAF, and NRAS is a molecule that inhibits all of the intermediates in the CREB pathway; the ultimate combination therapy. By selecting CREB as the target of inhibition, the problem of drug resistance in pathways involving cKIT, BRAF, and NRAS mutations may be overcome. In addition, efficacy of CREB pathway

inhibition may be increased, as direct inhibition of a transcription factor is more effective than indirect inhibition via intermediate proteins. Inhibition of CREB transcription factors leads to a decrease in cell proliferation by reducing CDK4/Cyclin D1 transcripts. (Swick et al., 2012). Inhibition of CREB also leads to apoptosis of affected cells via a number of mechanisms, notably absence of a transcription product of CREB, microphthalmia-associated transcription factor (MITF). MITF is vital for the expression of B-cell lymphoma 2 (BCL2), an anti-apoptotic regulatory protein. Overexpression of BCL2 leads to immortality of the cancer cell, while reduction of BCL2 induces apoptosis via caspase cascade (Mavromatis et al., 2004). By this mechanism, destruction of cancer cells using CREB inhibitors can be achieved. CREB inhibition may also lead to unwanted side effects. Due to the nature of CREB as a vital transcription factor, off-target cell deaths may be unavoidable. However, since most current melanoma therapies already act to inhibit CREB indirectly by binding to proteins such as cKIT, BRAF, and NRAS, a point of balance in therapy concentration can likely be reached whereby the therapy destroys tumor cells while sparing normal cells. The mechanisms for specifically targeting tumor cells, and methods of CREB inhibition, are discussed in the following section.

Methods of Inhibition and Targeting: Alex Wesselhoeft, Silas Chan

The simplest method of inhibiting an intracellular protein is using a small or large molecule to directly bind to the active site of the target. In this case, the molecule to be used would need to bind selectively to CREBs DNA binding domain, known as the cAMP response element. The sequence of the cAMP response element is known: 5 TTGACGTCA 3 (Schumacher et al., 2000). It thus becomes a simple task to inhibit CREB binding a free cAMP response element (so that its binding does not lead to gene transcription) to CREB would effectively halt CREBs activity through competitive inhibition. Several molecular possibilities exist for competitively inhibiting CREB; these possibilities include small molecules, proteinaceous large molecules, and DNA aptamers. DNA aptamers small single stands of DNA that selectively bind to a target molecule are perhaps the most promising (Zhu et al., 2012). Aptamers have been used to bind to several targets in cancer therapies, including vascular endothelial growth factor (VEGF), a signaling protein involved in angiogenesis that is upregulated in many tumors. These aptamers have been found to exhibit no immunogenicity (Ng et al., 2006). In addition, they can be engineered to selectively bind with high affinity to a target molecule. (Yangyuoru et al., 2012). Due to their properties of low immunogenicity and potential for high selectivity and affinity in binding, aptamers are wellsuited for a CREB-inhibition model of cancer therapy. In most cases, aptamers are screened through a process dubbed SELEX, whereby target molecules are washed in fragments of DNA and binding is retroactively determined (Ulrich et al., 2006). This process is used when binding targets for aptamers are unknown because of the complexity of binding interactions. However, an easily recognizable aptamer binding site is

present in CREB due to the known binding sequence of its DNA binding domain, cAMP response element. An aptamer with a sequence analogous to the cAMP response element may be able to bind to the DNA binding domain of CREB, thus inhibiting excessive transcription and removing the need for SELEX-based aptamer selection. Because CREB remains in the nucleus regardless of activation state, it is important that a cAMP response element aptamer be localized to the nucleus where it can bind to and inhibit the activity of CREB. In order to accomplish this, a nuclear localization sequence, such as the PKKKRKV amino acid sequence found in the SV40 virus (Kalderon et al., 1984), may be covalently bonded to the aptamer (Bremner et al., 2003; Hebert, 2003). These localization sequences guide proteins (or in this case, DNA molecules) into the nucleus by binding to importins, which then ferry the molecules through nuclear porins (Newmeyer et al., 1988). Due to nuclease degradation and small molecular weight, aptamers are cleared from the body relatively quickly; however, conjugation to molecules such as polyethylene glycol significantly increase half-life (Binkowski et al., 2005). It stands to reason that conjugation of a nuclear localization signal to an aptamer would also increase its half-life, thus increasing the potency of the therapy and retention of the therapy by cancerous cells. Entry into the cell is required before a nuclear localization sequence-bound aptamer can be transported to the nucleus to inhibit CREB. Nanospheres, which have been found to be efficient and capable of diffusing and passing through blood vessels to target deep tissue (Conti et al., 2006), present a unique opportunity for aptamer/protein conjugate transport into the cytosol of cancerous cells. Dendritic polymers (tree-shaped molecules, also known as dendrons) can be used to create globular nanoparticles encapsulating a molecule such as an aptamer for transport and timed release (She et al., 2013). Dendrons self-assemble into spherical particles

that are more efficient at transporting conjugated material into cells than non-assembled dendrons (Bernard et al., 2011). As the dendrons assemble, conjugated molecules become encapsulated within the nanospheres, which degrade over time and release their contents. PEGylation (conjugation of polyethylene glycol to another molecule) of the nanosphere surface increases water solubility and masks the nanoparticles from the immune system, thus reducing immunogenicity while simultaneously increasing survival of the therapy (Veronese et al., 2005). PEGylation of one tail of a dendron via click reactions in addition to conjugation of a nuclear localization sequence-bound aptamer to another tail of the dendron via hydrazone bonding could yield soluble non-immunogenic nanospheres encapsulating a CREB-inhibition therapy. Several cancer-specific targeting mechanisms exist inherently in the described methods of aptamer delivery. Nanoparticles formed with dendritic polymers degrade at higher rates in acidic conditions, increasing therapy release. Acidic environments are often found in tissues surrounding tumor cells (Robey, 2012), and melanoma is no exception (Bellone et al., 2013). Because of this, therapy concentrations will be larger at tumor sites even when nanoparticles are administered systemically. The EPR effect (enhanced permeability and retention) basic to all cancers ensures that tumor cells will obtain and retain more nanoparticles, and thus more therapy, than non-cancerous cells (Maeda, 2012). The EPR effect states that cancerous cells accumulate macromolecules faster than normal cells. Tumors must build their own blood supplies, which display abnormally wide fenestrations that allow macromolecules to pass at will. In addition, lymphatic drainage systems are not in place for efficient removal of material from the tumor environment (Matsumara et al., 1986). Indeed, it has been found that the EPR effect efficiently targets therapies to cancer cells when using only nanospheres conjugated with doxorubicin with no

visible toxicity (She et al., 2013). Because the proposed therapy utilizes a macromolecular (aptamer/nuclear localization signal) inhibitor of CREB in addition to delivery using nanoparticles, the EPR effect will be doubly active. Several proteins populate the CREB family. In addition to eight CREB subtypes, nonrelated proteins such as CREM and ATF-1 are also able to bind to the cAMP response element, and thus may be inhibited by a cAMP response element aptamer (Meyer et al., 1992). This phenomenon allows for concentration-based targeting; with CREB increased in both activity and number in only cancerous cells, the relative chance for an introduced aptamer to bind to CREB instead of another nuclear protein increases greatly. And due to the prevalence of non-CREB aptamer binding targets in normal cells, the likelihood of apoptosis due to CREB inhibition decreases greatly. Finally, a possible method that could be used to enhance targeting in cancers wherein mutated membrane receptors exist and are known is the modulation of nanosphere surface polymers. Polymers that bind to commonly mutated receptors such as cKIT could be conjugated with dendrons with a method similar to PEGylation. Dasatinib, a tyrosine kinase receptor inhibitor, is one such candidate (Das et al., 2006). While these mechanisms for specifically targeting cancerous cells are likely to reduce off-target effects, they may not be adequate. Indeed, direct inhibition of CREB could lead to side effects typical of most chemotherapies such as hair loss, fatigue, and nausea, all due to the cytotoxic effects of the therapy (Love et al., 1989). In order to decrease potential side effects, the nanoparticle-encapsulated aptamers can be incorporated into electrospun fibers. Electrospun fibers are materials woven from a mix of polymers, with different solutions used to hold the nanoparticle materials to deliver anticancer therapies (Huang et al., 2012). The

electrospun materials can be designed to contain a lipid-soluble solution to allow for easy binding of the nanoparticles, which utilize a lipid-soluble coating to deliver the anticancer therapies. This lipid solubility will allow the nanoparticles to diffuse deep into tissues while bypassing the vasculature. These fibers are crucial to the delivery of the therapy, since they can be modified in a way that will allow the therapy to be released at a consistent rate over an extended period of time. Consistent low-dose release of therapy will result in an even greater EPR effect, as normal cells will be able to balance the influx of therapy with the outflux of therapy, while cancerous cells will be unable to remove therapy from their cytosol due to lack of lymph vessels. Over time, CREB inhibition will remain constant in normal cells, and will rise in cancerous cells, eventually leading to apoptosis. In addition, these fibers can be biodegradable. The biodegradable aspect of electrospun fibers allows for the possibility of implantation of the fibers into internal tissues without need for eventual removal. The gradual, natural decomposition of the fibers makes it an ideal method for delivery of the therapy at the site of tumor development. These fibers, once coated with therapy, can be implanted into the site of the malignant melanoma. Once implanted in the site, gradual release of the drugs will occur due to the decomposition of the fibers. In addition, they can also be applied topically to the surface of the visible melanoma. In this case, it is not necessary to use biodegradable nanofibers, since topical application results in ease of removal. The therapy would be delivered to the site of ALM directly. The protocol designed by Karthikeyan et al. yielded uniform concentrations of nanoparticles attached to electrospun nanofibers. Because of this, this protocol can be utilized in the processing and formation of electrospun nanofibers for this treatment. That means a solution of aceclofenac/zein and pantoprazole/eudragit S 100, polymer solution, were combined in a 1:5

ratio to prepare for electrospinning. These can then be placed in 5 ml syringes and fed out at a rate of 1ml/h, while applying 25kV to the needle to start the propulsion. The polymer will then be collected on aluminum foil and dried (Karthikeyan et al., 2012). In our case, the same polymer solution will be utilized (zein and eudragit). Based on Karthikeyans results, consistent release of aceclofenac and pantoprazole was measured, in addition to the uniformity of the nanofibers. Using this method, the aptamer can first be encapsulated by nanoparticles and then merged with composite nanofibers to undergo electrospinning. Thus, a patch of nanofibers can be formed, while keeping the therapy intact.

Measuring Treatment Efficacy: Silas Chan, Alex Wesselhoeft

Several methods exist for measuring the efficacy of cancer treatments. A visual examination of the tumor site is often the first measure of effect. This is especially possible in melanomas, and often more so in ALM due to the location and drastic phenotypic shifts from normal skin that tumors display. Decrease or maintenance of tumor size can be physically measured by length and width. Assessment of all volar areas will also be necessary, since ALMs are aggressive and can spread quickly. Biopsies of tumor tissue and surrounding apparently healthy tissue can be obtained to observe progress of the cancer and invasion of surrounding tissues, in addition to changes in depth of the tumor. Tissue samples can be observed under a microscope to detect changes in tumor cell morphology that might suggest metastasis, such as mobility or lack of adhesive characteristics (Weeraratna et al., 2002). Tissue samples from the localized tumor and surrounding cells can be used to determine gene and protein expression using western blot and mRNA quantification techniques. Because CREB is more plentiful in most ALMs, CREB mRNA will also be more plentiful. Measuring quantity of CREB mRNA can therefore determine whether or not the inhibition of CREB by the aptamer is successful. CREB mRNA levels can also be used to determine the need for an increase in therapy quantity and concentration. To more accurately determine the efficacy of a CREB-binding aptamer and the nanoparticle/electrospun fiber targeting methods, protein and mRNA levels of CREB transcription products, such as CDK4 and Cyclin D1, can also be measured via western blot or

mRNA quantification. A relative decrease of these proteins or mRNAs in comparison to normal cells would indicate a successful treatment. Analysis of cell necrosis and apoptosis can be achieved using flow cytometry (Darzynkiewicz et al., 1997). Rates of death in both healthy and cancerous cells should be measured in order to both detect the occurrence of off-target effects of CREB inhibition, and measure the efficacy of the aptamer in forcing apoptosis in cancerous cells. Outcomes of these measurements can determine the need for a different concentration of therapy and the success or failure of the therapy. Progress can be updated on a weekly or monthly basis depending on the initial observed rate of change in tumor size (National Cancer Institute, 2010).

Quantitative Analysis of Treatment Concentration: Jorge Vargas, Alex Wesselhoeft

The proper concentrations of the drug will be figured first in vitro with ALM cells. This will be done through a MTT assay using WST dye to quantify cellular metabolism rates, and thus rates of cellular proliferation and numbers of viable cells. A cell experiences oxidation and reduction throughout its life cycle. Proliferating cells with high enzymatic activities would turn the dye to a dark color while inactive cells remain clear (Berridge et al., 2005). Therapy can be added to ALM cultures and reduction in metabolism/proliferation (reflected in changes in dye color) can be observed. Once the dye is the same shade as control non-cancerous cells tested using the same method, an inhibitory concentration has been achieved. A toxic concentration of therapy can also be measured with the same assay, instead determining the concentration of therapy that shows no color change. Control assays can be present in the form of non-cancerous epithelial cells. Required tissue concentrations for inhibition ranging from 0.5g/mL to 250g/mL can be expected, as most common chemotherapies fall into this broad range (Haouala et al. 2009). Normal epithelial cells would be bathed in the ideal concentrations of therapy yielded by ALM culture assays in order to determine off-target toxicity. However, since the EPR effect is mostly responsible for the targeting of the proposed therapy to tumor cells, a combined assay would be more informative; recreating a tumor embedded within normal epithelial tissue to observe the relative effects of therapy administration would better approximate actual desired concentrations. Time of inhibition/toxicity is also an important factor to consider when using nanospheres. Timed release can be measured by placing aptamer-nanoparticle conjugates in an aptamer-free media of varying acidity. Aptamer concentration in the media after varying times

can be assessed via spectrophotometry (Zhang et al., 2010). The determined rate of therapy release from the nanospheres will approximate the efficacy of nanospheres in maintaining a cyotoxic or cytostatic concentration in tumor cells, and exhibit the need for higher or lower concentrations of therapy. Variety in acidity of media will provide a good relative comparison of tumor and non-tumor environment release rates. Once we have figured the concentrations that cause inhibition or toxicity of ALM cells in vitro, progression to in vivo testing in mice would be the next necessary step. Therapy can be tested in both systemic and electrospun fiber-localized applications. In both cases, systemic drug concentrations can be measured by taking blood samples and tissue samples in animal models introduced to a carcinogen such as DMBA to spur ALM formation (Miyata et al., 2001). In human models, elastic-scattering spectroscopy (ESS) can be used to determine therapy concentrations without an invasive procedure (Mourant et al., 1999). Calibration curves measuring tissue/blood concentration against administration of varying quantities of therapy can show ideal levels of therapy administration needed to the achieve tissue accumulation levels deemed to be necessary with the MTT assay previously described (Haouala et al., 2009). Excretion rates of therapy can be measured by isolating aptamer sequences from urine, and used for discerning the possible rate of off-target cytotoxicity (Brody et al., 2000). Finally, toxicity of therapy concentrations to normal cells in vivo will present a more complete analysis of the efficacy of the EPR effect, in addition to other targeting mechanisms previously described. This toxicity can be measured again by MTT assay after removal of normal and cancerous cells from mice, and also by quantification of liver toxicity enzymes such as blood urea nitrate in normal mice (Kosem et al., 2012). Organs can be weighed after sacrifice to determine loss of tissue, and weight, behavior and phenotype changes of mice can be observed during treatment.

With therapy administration concentrations concluded in mice, volunteer trials would be necessary to ascertain whether or not the results of murine testing are valid for humans. Similar techniques can be used to determine efficacy of the therapy and toxic side effects; ESS to determine tissue concentrations of therapy, biopsies of tumor cells and western blots/RNA quantification to determine CREB inhibition, and visual approximations of ALM tumor regression. Toxicity can be measured by presence of physical side effects in addition to quantification of liver enzymes correlated with toxicity.

Future Directions: Muska Hassan

After using DNA aptamers and electrospun fibers to target CREB and inhibit cell proliferation, we can use the described in vivo and in vitro experiments to ascertain the effectiveness of this therapy. If our data suggest that the drug therapy has stopped the upregulation of CREB in cancerous melanoma cells, we can look into the use of CREB inhibition in breast cancer, leukemia, and neuroblastoma, which also show changes in CREB concentration in cancer cells. We can also try to apply our method of treating primary tumors by inhibiting other transcription factors that leads to cell proliferation in cancer cells such as STAT3, 5, NFB, -catenin, Notch, c-JUN and GLi (Darnell, 2002). By using such methods, we can accelerate the personalization of medicine for the treatment of various types of cancers. If our in-vitro and in vivo experiments suggest that despite inhibiting CREB, we were not successful at inhibiting cell proliferation in cancer cells, we can try to manipulate the dosage concentration of the therapy. It may be that CREB is not the only transcription factor in ALM that when changed in terms of activity causes continuous cell proliferation. There may be other transcription factors that are also responsible for cell proliferation. We can look more into cancer cell signaling pathways and try to identify those transcription factors that contribute to cell proliferation and target it the same way we would with CREB by using a DNA aptamer encapsulated in nanospheres and delivered with electrospun fibers. Identification of other transcription factors can also help us with targeting other cancer types.

Citations:

1. Adam, Stephan A., Rachel Sterne-Marr, and Larry Gerace. "Nuclear Protein Import Using Digitonin-permeabilized Cells." Methods in Enzymology 219 (1992): 97110. Sciencedirect.com. Reconstitution of Intracellular Transport. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/007668799219013V>. 2. "Adjuvant Therapy." NCI Dictionary of Cancer Terms. National Cancer Institute. Web. 11 Feb. 2013. <http://www.cancer.gov/dictionary?cdrid=45587>. 3. Amiri, Payman, Mina Aikawa, Jeff Dove, Darrin D. Stuart, Daniel Poon, Teresa Pick, Savithri Ramurthy, Sharadha Subramanian, Barry Levine, Abran Costales, Alex Harris, and Renhow Paul. "CHIR-265 Is a Potent Selective Inhibitor of C-Raf/B-Raf/mutB-Raf That Effectively Inhibits Proliferation and Survival of Cancer Cell Lines with Ras/Raf Pathway Mutations -- Amiri Et Al. 2006 (1): 1140 -- AACR Meeting Abstracts." Proc Amer Assoc Cancer Res 47 (2006). Experimental and Molecular Therapeutics 40: Cytoplasmic Signaling Inhibitors. Chiron Corp. Web. 11 Feb. 2013. <http://www.aacrmeetingabstracts.org/cgi/content/abstract/2006/1/1140-a>. 4. Bellone, Matteo, Arianna Calcinotto, Paola Filipazzi, Angelo De Milito, Stefano Fais, and Licia Rivoltini. The Acidity of the Tumor Microenvironment Is a Mechanism of Immune Escape That Can Be Overcome by Proton Pump Inhibitors Landes Bioscience Journals: OncoImmunology, Jan. 2013. Web. 19 Feb. 2013. 5. Bennett, D. R., D. Wasson, J. D. MacArthur, and M. A. McMillen. "The Effect of Misdiagnosis and Delay in Diagnosis on Clinical Outcome in Melanomas of the Foot." J American College of Surgery 179.3 (1994): 279-84. National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/8069422>. 6. Bernard, Anna, Paola Posocco, Sabrina Pricl, Marcelo Calderon, Rainer Haag, Mark E. Hwang, Victor W. T. Shum, Daniel W. Pack, and David K. Smith. "Degradable SelfAssembling Dendrons for Gene Delivery: Experimental and Theoretical Insights into the Barriers to Cellular Uptake." Journal of the American Chemical Society 133.50 (2011): 20288-0300. Pubs.acs.org. Department of Chemistry, University of York; Molecular Simulation Engineering Laboratory, Department of Industrial Engineering and Information Technology; Institut Fur Chemie Und Biochemie, Freie Universitat Berlin; Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign; American Chemical Society, 31 Oct. 2011. Web. 18 Feb. 2013. <http://pubs.acs.org/doi/abs/10.1021%2Fja2070736>. 7. Berridge, M. V., P. M. Herst, and A. S. Tan. "Tetrazolium Dyes as Tools in Cell Biology: New Insights into Their Cellular Reduction." National Center for Biotechnology Information. Biotechnology Annual Review, 2005. Web. 19 Feb. 2013. 8. Binkowski, B., R. Miller, and P. Belshaw. "Ligand-Regulated Peptides: A General Approach for Modulating Protein-Peptide Interactions with Small Molecules." Chemistry & Biology 12.7 (2005): 847-55. Pubmed.gov. US National Library of Medicine; National Institutes of Health. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/16039531>. 9. Bradford, Porcia T., MD, Alisa M. Goldstein, PhD, Mary L. McMaster, MD, and Margaret A. Tucker, MD. "Acral Lentiginous Melanoma: Incidence and Survival Patterns in the

United States, 1986-2005." Arch Dermatology 145.4 (2009): 42734.Archderm.jamanetwork.com. Web. 16 Feb. 2012. <http://archderm.jamanetwork.com/article.aspx?articleid=712002>. 10. Bremner, K. Helen, Leonard W. Seymour, Ann Logan, and Martin L. Read. "Factors Influencing the Ability of Nuclear Localization Sequence Peptides To Enhance Nonviral Gene Delivery." Bioconjugate Chemistry 15.1 (2004): 152-61. Pubs.acs.org. American Chemical Society; Cancer Research UK Institute for Cancer Studeis, University of Birmingham; Department of Clinical Pharmacology, University of Oxford; Department of Medicine, University of Birmingham, 13 Dec. 2003. Web. 18 Feb. 2013. <http://pubs.acs.org/doi/abs/10.1021/bc034140k>. 11. Brody, Edward N., and Larry Gold. "Aptamers as Therapeutic and Diagnostic Agents."ScienceDirect.com. Reviews in Molecular Biotechnology, 1 Mar. 2000. Web. 19 Feb. 2013. 12. Conti, M., Tazzari, V., Baccini, C., Pertici, G., Serino, L. P., & Giorgi, U. D. (2006). Anticancer Drug Delivery with Nanoparticles. In Vivo, 20(6A), 697701. 13. Curtin, J. A., K. Busam, D. Pinkel, and B. C. Bastian. "Somatic Activation of KIT in Distinct Subtypes of Melanoma." Journal of Clinical Oncology 24.26 (2006): 4340-346. Journal of Clinical Oncology. American Society of Clinical Oncology, 14 Aug. 2006. Web. 11 Feb. 2013. <http://jco.ascopubs.org/content/24/26/4340.long>. 14. Darnell, J. E. "Transcription Factors as Targets for Cancer Therapy." National Center for Biotechnology Information. U.S. National Library of Medicine, 2 Oct. 2002. Web. 19 Feb. 2013. 15. Darzynkiewicz, Z., G. Juan, X. Li, W. Gorczyca, T. Murakami, and F. Traganos. "Cytometry in Cell Necrobiology: Analysis of Apoptosis and Accidental Cell Death (necrosis)."National Center for Biotechnology Information. U.S. National Library of Medicine, 1 Jan. 1997. Web. 19 Feb. 2013. 16. Das, J., P. Chen, D. Norris, R. Padmanabha, J. Lin, RV Moquin, Z. Shen, LS Cook, AM Doweyko, S. Pitt, S. Pang, Dr Shen, Q. Fang, HF De Fex, KW McIntyre, DJ Shuster, KM Gillooly, K. Behnia, GL Schieven, J. Wityak, and JC Barrish. "2-aminothiazole as a Novel Kinase Inhibitor Template. Structure-activity Relationship Studies toward the Discovery of N-(2-chloro-6-methylphenyl)-2[[6-[4-(2-hydroxyethly)-1-piperazinyl)]-2methyl-4pyrimidinyl]amino)-1,3-thiazole-5-carboxamide (dasatinib, BMS-254825) as a Potent PanSrc Kinase Inhibitor." J Med Chem 49.23 (2006): 6819-832. Pubmed.gov. US National Library of Medicine; National Institutes of Health; Bristol-Myers Squibb Pharmaceutical Research Insititute. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/17154512>. 17. El-Telbany, A., and PC MA. "Cancer Genes in Lung Cancer: Racial Disparities: Are There Any?" Genes Cancer 3.7-8 (2012): 467-80. Pubmed.gov. US National Library of Medicine; National Institutes of Health. Web. 15 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/23264847>. 18. "Follow-up Care After Cancer Treatment." National Cancer Institute Factsheet. National Cancer Institute, 17 May 2010. 19. Fisher, Rosalie, and James Larkin. "Vemurafenib: A New Treatment for BRAF-V600 Mutated Advanced Melanoma." Cancer Managment Resources 4 (2012): 243-52. National Institutes of Health. US National Library of Medicine, 8 Aug. 2012. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3421463/>.

20. Franke, W., N. Neumann, T. Ruzicka, and K. Schulte. "Plantar Malignant Melanomaa Challenge for Early Recognition." Melanoma Research December 10.6 (2000): 57176. Pubmed.gov. National Institute of Health, Department of Dermatology , Heinrich-Heine University. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/11198479>. 21. Haflidadttir, Benedikta S., Kristn Bergsteinsdttir, Christian Praetorius, and Eirkur Steingrmsson. "MiR-148 Regulates Mitf in Melanoma Cells." Ed. Dong-Yan Jin. PLoS ONE 5.7 (2010): E11574. Plosone.org. Web. 13 Feb. 2013. <http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0011574>. 22. Haouala, A, et al. "Therapeutic Drug Monitoring of the New Targeted Anticancer Agents Imatinib, Nilotinib, Dasatinib, Sunitinib, Sorafenib and Lapatinib by LC Tandem Mass Spectrometry." ScienceDirect.com. Journal of Chromatography B, 15 July 2009. Web. 19 Feb. 2013. 23. Hauschild, Axel, Sanjiv S. Agarwala, Uwe Trefzer, David Hogg, Caroline Robert, Peter Hersey, Alexander Eggermont, Stephan Grabbe, Rene Gonzalez, Jens Gille, Christian Peschel, Dirk Schadendorf, Claus Garbe, Steven O'Day, Adil Daud, J. Michael White, Chenghua Xia, John M. Kirkwood, and Ulrich Keilholz. "Results of a Phase III, Randomized, Placebo-Controlled Study of Sorafenib in Combination With Carboplatin and Paclitaxel As Second-Line Treatment in Patients With Unresectable Stage III or Stage IV Melanoma." Journal of Clinical Oncology 27.17 (2009): 2823-830. Jco.ascopubs.org. American Society of Clinical Oncology, 06 Apr. 2009. Web. 11 Feb. 2013. <http://jco.ascopubs.org/content/27/17/2823.full.pdf>. 24. Hebert, E. "Improvement of Exogenous DNA Nuclear Importation by Nuclear Localization Signal-bearing Vectors: A Promising Way for Non-viral Gene Therapy?" Biology of the Cell 95.2 (2003): 59-68. Biocellusa.org. Science Direct; Vectorologie Et Trafic Intracellulaire, Centre De Biophysique Moleculaire. Web. 18 Feb. 2013. <http://www.biolcellusa.org/boc/095/0059/boc0950059.pdf>. 25. Heidorn, Sonja J., Carla Milagre, Steven Whittaker, Arnaud Nourry, Ion Niculescu-Duvas, Nathalie Dhomen, Jahan Hussain, Jorge S. Reis-Filho, Caroline J. Springer, Catrin Pritchard, and Richard Marais. "Kinase-Dead BRAF and Oncogenic RAS Cooperate to Drive Tumor Progression through CRAF." Cell 4.2 (2010): 209-21. Cell.com. The Brothers RAF. Web. 11 Feb. 2013. <http://www.cell.com/retrieve/pii/S0092867409016262>. 26. Henderson, Y. C., Y. Chen, M. J. Frederick, S. Y. Lai, and G. L. Clayman. "MEK Inhibitor PD0325901 Significantly Reduces the Growth of Papillary Thyroid Carcinoma Cells In Vitro and In Vivo." Molecular Cancer Therapeutics 9.7 (2010): 1968-976.National Institutes of Health. US National Library of Medicine, Department of Head and Neck Surgery, The University of Texas, 29 June 2010. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/20587665>. 27. Huang, C., Soenen, S. J., Rejman, J., Trekker, J., Chengxun, L., Lagae, L., De Smedt, S. C. (2012). Magnetic Electrospun Fibers for Cancer Therapy. Advanced Functional Materials, 22(12), 24792486. doi:10.1002/adfm.201102171 28. Impey, S., S. Mccorkle, H. Chamolstad, J. Dwyer, G. Yochum, J. Boss, S. Mcweeney, J. Dunn, G. Mandel, and R. Goodman. "Defining the CREB RegulonA Genome-Wide Analysis of Transcription Factor Regulatory Regions." Cell 119.7 (2004): 1041054.Sciencedirect.com. Vollum Institute, Oregon Health and Science University; Biology Department, Brookhaven National Laboratory; Department of Public Health and Preventative Medicine, Oregon Health and Science University; Deparment of Microbiology and

Immunology, Emory School of Medicine; Howard Hughes Medical Institute, Department of Neurobiology and Behavior, The State University of New York at Stony Brook, Cell Press. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/S0092867404011596>. 29. Ives, N. J., R. L. Stowe, P. Lorigan, and K. Wheatley. "Chemotherapy Compared With Biochemotherapy for the Treatment of Metastatic Melanoma: A Meta-Analysis of 18 Trials Involving 2,621 Patients." Journal of Clinical Oncology 25.34 (2007): 5426-434. Journal of Clinical Oncology. American Society of Clinical Oncology. Web. 11 Feb. 2013. <http://jco.ascopubs.org/content/25/34/5426.full.pdf>. 30. Jancic, Dragana, Mikel Lopez De Armentia, Luis M. Valor, Roman Olivares, and Angel Barco. "Inhibition of CAMP Response Element-Binding Protein Reduces Neuronal Excitability and Plasticity, and Triggers Neurodegeneration." Cerebral Cortex 19.11 (2009): 2535-547. Oxfordjournals.org. 12 Feb. 2009. Web. 13 Feb. 2013. <http://cercor.oxfordjournals.org/content/19/11/2535.full>. 31. Kageshita, T., Nakamura, T., Yamada, M., Kuriya, N., Arao, T., & Ferrone, S. (1991). Differential Expression of Melanoma Associated Antigens in Acral Lentiginous Melanoma and in Nodular Melanoma Lesions. Cancer Research, 51(6), 17261732. 32. Kalderon, Daniel, Bruce L. Roberts, William D. Richardson, and Alan E. Smith. "A Short Amino Acid Sequence Able to Specify Nuclear Location." Cell 39.3 (1984): 499509. Pubmed.gov. SciVerse; Biochemistry Division National Insitute for Medical Research. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/0092867484904574>. 33. Karthikeyan, K., Guhathakarta, S., Rajaram, R., & Korrapati, P. S. (2012). Electrospun zein/eudragit nanofibers based dual drug delivery system for the simultaneous delivery of aceclofenac and pantoprazole. International Journal of Pharmaceutics, 438(12), 117122. doi:10.1016/j.ijpharm.2012.07.075 34. Kosem, Nuttavut, Kazuhiro Ichikawa, Hideo Utsumi, and Primchanien Moongkarndi. "In Vivo Toxicity and Antitumor Activity of Mangosteen Extract." Journal of Natural Medicines, 01 May 2012. Web. 19 Feb. 2013. 35. Love, Richard R., Howard Leventhal, Douglas V. Easterling, and David R. Nerenz. "Side Effects and Emotional Distress during Cancer Chemotherapy." Cancer 63.3 (1989): 60412. Onlinelibrary.wiley.com. 29 June 2006. Web. 18 Feb. 2013. <http://onlinelibrary.wiley.com/doi/10.1002/1097-0142(19890201)63:3%3C604::AIDCNCR2820630334%3E3.0.CO;2-2/abstract>. 36. Maeda, H. "Macromolecular Therapeutics in Cancer Treatment: The EPR Effect and beyond." J Control Release 164.2 (2012): 138-44. Pubmed.gov. US National Library of Medicine; National Institutes of Health; Institute of DDS Research. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/22595146>. 37. Maira, S.-M., F. Stauffer, J. Brueggen, P. Furet, C. Schnell, C. Fritsch, S. Brachmann, P. Chene, A. De Pover, K. Schoemaker, D. Fabbro, D. Gabriel, M. Simonen, L. Murphy, P. Finan, W. Sellers, and C. Garcia-Echeverria. "Identification and Characterization of NVPBEZ235, a New Orally Available Dual Phosphatidylinositol 3-kinase/mammalian Target of Rapamycin Inhibitor with Potent in Vivo Antitumor Activity." Molecular Cancer Therapeutics 7.7 (2008): 1851-863. Print. 38. Matsumura, Yasuhiro, and Hiroshi Maeda. "A New Concept for Macromolecular Therapeutics in Cancer Chemotherapy: Mechanism of Tumoritropic Accumulation of Proteins and the Antitumor Agent Smancs." The Journal of Cancer Research 46

(1986).Aacrjournals.org. Web. 18 Feb. 2013. <http://cancerres.aacrjournals.org/content/46/12_Part_1/6387.abstract>. 39. Mavromatis, Blanche H., and Bruce D. Cheson. "Novel Therapies for Chronic Lymphocytic Leukemia." Blood Reviews 18.2 (2004): 137-48. ScienceDirect. Lombard Cancer Center, Georgetown University Medical Center; Blood Reviews. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/S0268960X03000390>. 40. "Melanoma Treatment (PDQ)." National Cancer Institute. National Institutes of Health, 26 Oct. 2012. Web. 11 Feb. 2013. <http://www.cancer.gov/cancertopics/pdq/treatment/melanoma/HealthProfessional/page4#Re ference4.1>. 41. Meyer, Terry E., and Joel F. Habener. "Cyclic AMP Response Element Binding Protein CREB and Modulator Protein CREM Are Products of Distinct Genes." Nucleic Acids Research 20.22 (1992): 6106. Pubmed.gov. US National Library of Medicine; National Institutes of Health. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC334485/>. 42. Microfabricated electrospun collagen membranes for 3-D cancer models and drug screening applications. (n.d.). Retrieved February 11, 2013, from http://www.academia.edu/885880/Microfabricated_electrospun_collagen_membranes_for_3D_cancer_models_and_drug_screening_applications 43. Miyata, M., M. Furukawa, K. Takahashi, F. J. Gonzalez, and Y. Yamazoe. "Mechanism of 7,12-dimethylbenz[a]anthracene-induced Immunotoxicity: Role of Metabolic Activation at the Target Organ." National Center for Biotechnology Information. Japanese Journal of Pharmacology, July 2001. Web. 19 Feb. 2013. 44. Mourant, Judith R., Tamara M. Johnson, Gerrit Los, and Irving J. Bigio. "Non-invasive Measurement of Chemotherapy Drug Concentrations in Tissue: Preliminary Demonstrations of in Vivo Measurements." IOP Science. Physics in Medicine and Biology, 1999. Web. 19 Feb. 2013. 45. "Non-invasive Measurement of Chemotherapy Drug Concentrations in Tissue: Preliminary Demonstrations of in Vivo Measurements." IOP Science. Physics in Medicine and Biology, 1999. Web. 19 Feb. 2013. 46. Newmeyer, Donald D., and Douglass J. Forbes. "Nuclear Import Can Be Separated into Distinct Steps in Vitro: Nuclear Pore Binding and Translocation." Cell 52.5 (1988): 64153. ScienceDirect. Department of Biology University of California at San Diego La Jola; Cell Press. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/0092867488904023>. 47. Ng, Eugene W. M., David T. Shima, Perry Calias, Emmett T. Cunningham, David R. Guyer, and Anthony P. Adamis. "Pegaptanib, a Targeted Anti-VEGF Aptamer for Ocular Vascular Disease." Nature Reviews Drug Discovery 5.2 (2006): 123-32.Nature.com. Pubmed.gov. Web. 18 Feb. 2013. <http://www.nature.com/nrd/journal/v5/n2/abs/nrd1955.html>. 48. Palmieri, Giuseppe, MariaElena Capone, MariaLibera Ascierto, Giusy Gentilcore, David F. Stroncek, Milena Casula, MariaCristina Sini, Marco Palla, Nicola Mozzillo, and Paolo A. Ascierto. "Main Roads to Melanoma." Journal of Translational Medicine 7.1 (2009): 86. Journal of Translational Medicine. Istituto Di Chimica Biomolecolare, Consiglio Nazionale Delle Ricerche; Istituto Nazionale Tumori; Cell Processing Section, Department of Transfusion Medicine Clinical Center, 14 Oct. 2009. Web. 13 Feb. 2013. <http://www.translational-medicine.com/content/7/1/86>.

49. Park, Hyun Sun, and Kwang Hyun Cho. "Acral Lentiginous Melanoma in Situ: A Diagnostic and Management Challenge." Cancers 2.2 (2010): 642-52. Mdpi.com. Department of Dermatology, Seoul National University College of Medicine, 20 Apr. 2010. Web. 17 Feb. 2013. <http://www.mdpi.com/2072-6694/2/2/642>. 50. Patterson, Karen. "Race, Genetics and Cancer." CureToday.com: Winter Supplement 2009 Article. CureToday.com, 12 Dec. 2009. Web. 11 Feb. 2013. <http://www.curetoday.com/index.cfm/fuseaction/article.show/id/2/article_id/1329>. 51. Processing of polymer nanofibers through electrospinning as drug delivery systems. (n.d.). Retrieved February 11, 2013, from http://www.academia.edu/1545586/Processing_of_polymer_nanofibers_through_electrospin ning_as_drug_delivery_systems 52. Puig-Butill, Jean A., Celia Badenas, Zighereda Ogbah, Cristina Carrera, Josep Malvehy, Paula Aguilera, and Susana Puig. "Genetic Alterations in RAS-regulated Pathway in Acral Lentiginous Melanoma." Experimental Dermatology February 22.2 (2013): 148-50. National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/23362874>. 53. Robey, I. "P01.30. The Role of PH in Cancer." BMC Complement Alternative Medicine 12.1 (2012): 30. Ncbi.nlm.nih.gov. Us National Library of Medicine; National Institutes of Health; BioMed Central, 12 June 2012. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3373632/>. 54. Sakamoto, K. M., and D. A. Frank. "CREB in the Pathophysiology of Cancer: Implications for Targeting Transcription Factors for Cancer Therapy." Clinical Cancer Research 15.8 (2009): 2583-587. Aacrjournals.org. Department of Medical Oncology, Dana-Farber Cancer Institute, 7 Apr. 2009. Web. 13 Feb. 2013. <http://clincancerres.aacrjournals.org/content/15/8/2583.full>. 55. Saldanha, G., L. Potter, P. DaForno, and J. H. Pringle. "Cutaneous Melanoma Subtypes Show Different BRAF and NRAS Mutation Frequencies." Clinical Cancer Research 12.15 (2006): 4499-505. Pubmed.gov. Department of Cancer Studies and Molecular Medicine, University of Leicester; US National Library of Medicine; National Institutes of Health. Web. 15 Feb. 2013. <www.ncbi.nim.nih.gov/pubmed/16899595>. 56. Schumacher, Maria A., Richard H. Goodman, and Richard G. Brennan. "The Structure of a CREB BZIPSomatostatin CRE Complex Reveals the Basis for Selective Dimerization and Divalent Cation-enhanced DNA Binding." The Journal of Biological Chemistry 45th ser. 275.November (2000): 35242-5247. Http://jcb.org. The American Society for Biochemistry and Molecular Biology, Inc., Department of Biochemistry and Molecular Biology, Vallum Institute, Oregon Health Sciences. Web. 11 Feb. 2013. <http://www.jbc.org/content/275/45/35242.full.pdf+html>. 57. She, Wenchuan, Kui Luo, Gang Wang, Yanyan Gengy, Li Li, Bin He, and Zhongwei Gu. "The Potential of Self-assembled, PH-responsive Nanoparticles of MPEGylated Peptide Dendron-doxorubicin Conjugates for Cancer Therapy." Biomaterials 34.5 (2013): 1613623. ScienceDirect. National Engineering Research Center for Biomaterials, Sichuan University. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/S0142961212012513>. 58. Sill, T. J., & Von Recum, H. A. (2008). Electrospinning: Applications in drug delivery and tissue engineering. Biomaterials, 29(13), 19892006. doi:10.1016/j.biomaterials.2008.01.011

59. Sosman JA, Kim KB, Schucter L, et al. Survival in BRAF V600-mutant advanced melanoma treated with vemurafenib. N Engl J Med 366 (8): 707-14, 2012 60. "A Study Of Oral PD-0332991, A Cyclin-Dependent Kinase Inhibitor, In Patients With Advanced Cancer." Clinicaltrials.gov. Pfizer. Web. 11 Feb. 2013. <http://clinicaltrials.gov/show/NCT00141297>. 61. Swick, Julie M., and John C. Maize Sr., MD. "Molecular Biology of Melanoma." Journal of the American Academy of Dermatology 67.5 (2012): 1049-054. Sciencedirect.com. Department of Dermatology, Medical University of South Carolina; DermPath Diagnostics of South Carolina. Web. 18 Feb. 2013. <http://www.sciencedirect.com/science/article/pii/S0190962212001624>. 62. Thakur, Das Meghna, Fernando Salangsang, Allison Landman, William R. Sellers, Nancy K. Pryer, Mitchell P. Levesque, Reinhard Dummer, Martin McMahon, and Darrin D. Stuart. "Modelling Vemurafenib Resistance in Melanoma Reveals a Strategy to Forestall Drug Resistance." Nature 494 (2013): 251-55. Nature.com. 09 Jan. 2013. Web. 18 Feb. 2013. <http://www.nature.com/nature/journal/v494/n7436/abs/nature11814.html>. 63. Tsao, Hensin, Lynda Chin, Levi Garraway, and David Fisher. "Melanoma: From Mutations to Medicine." Genes and Development 26 (2012). Genesdev.cship.org. Cold Spring Harbor Laboratory Press. Web. 13 Feb. 2013. <http://genesdev.cshlp.org/content/26/11/1131.full>. 64. Ulrich, Henning, Cleber A. Trujillo, Arthur A. Nery, Janaina M. Alves, Paromita Majumder, Rodrigo R. Resende, and Antonio H. Martins. "DNA and RNA Aptamers: From Tools for Basic Research Towards Therapeutic Applications." Combinatorial Chemistry & High Throughput Screening 9.8 (2006): 619-32. Pubmed.gov. US National Library of Medicine; National Institutes of Health; Departamento De Bioquimica, Instituto De Quimica, Universidade De Sao Paulo. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/17017882>. 65. Uong, Audrey, and Leonard I. Zon. "Melanocytes in Development and Cancer." Journal of Cellular Physiology 222.1 (2010): 38-41. Onlinelibrary.wiley.com. 30 Sept. 2009. Web. 14 Feb. 2013. <onlinelibrary.wiley.com.sil.library.umass.edu/doi/10.1002/jcp.21935/full>. 66. Veronese, F., and G. Pasut. "PEGylation, Successful Approach to Drug Delivery." Drug Discovery Today 10.21 (2005): 1451-458. Pubmed.gov. Drug Discovery Today; Department of Pharmaceutical Sciences, Padua University. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/16243265>. 67. Veronesi, Umberto, and Natale Casinelli, MD. "Narrow Excision (1cm-margin) A Safe Procedure for Thin Cutaneous Melanoma." Arch Surg 126.4 (1991): 438-41. Pubmed.gov. World Health Organization Melanoma Programme, National Tumor Institute. Web. 11 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/2009058>. 68. Weeraratna, Ashani T., Yuan Jiang, Galen Hostetter, Kevin Rosenblatt, Paul Duray, Michael Bittner, and Jeffrey Trent. "Wnt5a Signaling Directly Affects Cell Motility and Invasion of Metastatic Melanoma." Science Direct. Cancer Cell, 3 Apr. 2002. Web. 18 Feb. 2013. 69. Xiao, X., B.X. Li, B. Mitton, A. Ikeda, and K.M. Sakamoto. "Targeting CREB for Cancer Therapy: Friend or Foe." Current Cancer Drug Targets 10.4 (2010): 384-91. Pubmed.gov. US National Library of Medicine; National Institutes of Health; Program in Chemical Biology, Oregon Health & Science University. Web. 18 Feb. 2013. <http://www.ncbi.nlm.nih.gov/pubmed/20370681>. 70. Yangyuoru, Philip M., Soma Dhakal, Zhongbo Yu, Deepak Koirala, Simon Mwongela, and Hanbin Mao. "Single-Molecule Measurements of the Binding between Small Molecules and

DNA Aptamers." Analytical Chemistry 84.12 (2012): 5298-303.Pubs.acs.org. School of Science and Technology, Georgia Gwinnett College, Department of Chemistry and Biochemistry, Kent State University; American Chemical Society, 23 May 2012. Web. 18 Feb. 2013. <http://pubs.acs.org/doi/abs/a/ac300427d>. 71. Yu, D.-G. (2009). Electrospun nanofiber-based drug delivery systems. Health, 01(02), 67 75. doi:10.4236/health.2009.12012 72. Zhang, Jing, Xing Jia, Xiao-Jing Lv, Yu-Lin Deng, and Hai-Yan Xie. "Fluorescent Quantum Dot-labeled Aptamer Bioprobes Specifically Targeting Mouse Liver Cancer Cells."ScienceDirect.com. Talanta, 15 Apr. 2010. Web. 19 Feb. 2013. 73. Zhu, Guizhi, Mao Ye, Michael J. Donovan, Erqun Song, Zilong Zhao, and Weihong Tan. "Nucleic Acid Aptamers: An Emerging Frontier in Cancer Therapy." Chemical Communications 48.85 (2012): 10472-0480. Rsc.org. RSC Publishing, Chemical Communications, 21 Aug. 2012. Web. 18 Feb. 2013. <http://pubs.rsc.org/en/content/articlelanding/2012/CC/c2cc35042d>.