Introduction outcomes that ultimately determine the fate

IntroductionReverse phase protein array (RPPA) is aproteomic assay designed to measure protein expression in a number of samples,simultaneously. Proteomics is the large scale study of a proteins’ structure,function and its interactions with other proteins. The study of proteins hasproven difficult compared to genomic studies. This is because a proteinsfunction, structure and post translational state fluctuates greatly from cellto cell and time to time. Post translational modifications (PTMs) such as phosphorylation,acetylation and ubiquitination, are responsible for determining whether aprotein is biologically active or not. Active proteins, such as phosphorylatedkinases, can ignite sequential phosphorylation of a number of proteins involvedin a particular pathway. These signaling pathways can have a wide range ofoutcomes that ultimately determine the fate of a cell- whether it lives ordies.

Thus, proteomic studies can be extremely useful in understandingdiseases, their progression and in developing targeted therapies. Although awide range of proteomic assays can determine whether a protein is present ornot, RPPA is one of the few that can examine the activation state of a protein.The activation state of a protein can prove vital when studying multistepdiseases such as cancer. A proteins activation state can have both genomic andepigenetic influences. Genomic analysis has proven key in revealing oncogenesand tumor suppressors whose protein products result in cancer cells immortality.

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Because it is the product of these genes, a deregulated protein, whichfacilitates the cellular effects seen in cancer, cancer research has nowshifted its focus to the cancer proteome. This proteomic research is aided byproteomic based assays such as RPPA (Boja & Rodriguez, 2014) .  In this review the RPPAprocess will be explored in depth with its advantages and disadvantageshighlighted. Its roles in cancer research will be demonstrated by example andits possible future shall be explored.

 BackgroundProtein microarrays are high throughput,robust arrays that allow for the characterization of multiple proteins fromseveral samples simultaneously (Reymond Sutandy, Qian, Chen, & Zhu, 2013). These proteomic arrays are essential in protein detection. Thereare three main types of protein arrays- sandwich type where the antigen istrapped between a primary and secondary antibody, antigen capture array wherethe antigen is hybridized to an informer molecule and reverse phases assays.

RPPAis similar to other immunoassays in that it uses primary antibodies to probe asample. Using antibodies to unique epitopes offers high sensitivity. Theprinciple is relatively simple. ‘Reverse phase’ refers to the analyte beingimmobilized to the solid surface.

A primary antibody (which must undergo astringent validation process) is then applied overnight in solution phase. Asecondary antibody, conjugated to an informer (e.g.HRP) is introduced. The secondaryantibody will recognize the primary, and once attached a signal can be detected(Liotta et al.

, 2003). Typical forward phase assays, such as ELISA, fix the primaryantibody to the solid surface. The sample is then applied in solution phase andincubated overnight.

This requires a lot of sample which is not viable for clinicaltrials whereby the sample can only be obtained via a biopsy. The minisculesample required by RPPA is a key advantage over forward phase arrays. In forwardphase, second primary antibody must also be applied to the sample to facilitatesecondary antibody binding. The specificity and cost of two primary antibodiesis another example of how RPPA supersedes forward phase arrays. RPPA allows for a large multitude ofsamples to be tested for specific protein/phosphoprotein under the same experimentalconditions, at the same time (Poetz et al., 2005).The general procedure can be visualized in figure 1.1.

The samples to be analyzed may be cellular lysates (Nishizuka et al., 2003), tissue samples  (Agarwal et al., 2009), oreven bodily fluids such as CSF, serum, urine etc. (Janzi et al., 2009). RPPA can also examine heterogeneous cell populations from tissuesamples (Akbani et al.

, 2014) – Cells grown in culture often have different protein profilescompared to cells in a human tissue due to the tissues micro-environment. Whendeveloping drug therapies this needs to be considered. The process of lasercapture microdissection has overcome this problem and can be used inconjunction with RPPA to obtain a sample that is an accurate representation ofa cells proteome (Bonner et al., 1997). It is still essential that once a tissue sample is obtained itmust be treated and preserved immediately so as to avoid any trauma or kinase,phosphoprotein alteration.

 Figure1.1 (Spurrier, Ramalingam, & Nishizuka, 2008) Protein extraction is carried out by celllysis using denaturing buffers and protease/phosphatase inhibitors. For highthroughput assays, the cell lysates obtained are generally serially diluted forquantification purposes as well as quality control. Roughly 1nL of lysate isthen printed onto a nitrocellulose glass slide via an automatic pin based microarrayer.Each slide is then probed with a primary antibody, followed by a secondary,which can be detected by fluorescent of chemiluminescent assays.

Software thenquantifies signal intensities for each spot. Central to RPPA preparation is antibodyscreening. Due to the required sensitivity of antibodies, all must firstundergo a strict validation process. Western blots (WB) are performed using thesame material as used in RPPA. Importantly, antibodies must display a singleband at the appropriate molecular weight for that protein. Antibodies againstphosphorylated proteins must show specificity against stimulated (growthfactors) or inhibited proteins (inhibitors). In many cases antibodyconcentration, compared to that used in WB, will be increased in RPPA.

Someantibodies may also need a longer incubation time. The limited number ofsensitive antibodies is RPPAs greatest disadvantage. Guidelines or methods forantibody validation are not available as of yet however a range of standardantibodies is available on the MD Anderson cancer webpage. One of the great advantages of RPPA is itsability to detect phopho-proteins or other PTM proteins.

This is essential indetermining the activation state of a protein. To do this the anylate issubjected to two different antibodies, one directed against the protein (e.g.p70S6K) and one aimed at the phosphorylated protein (e.g. p70 Ser-394).

Thedifference in levels between these two will allow one to determine theactivation state of the protein (Espina, Wulfkuhle, Calvert, Petricoin, & Liotta,2007). Onceobtained protein activation levels can be compared to different samples e.g.

activation of p70S6k in cancer or normal tissues.  DiscussionProteins are the functional units incells that achieve all biological processes such as cell migration,proliferation, apoptosis etc. Each cell has its own unique proteome that isreflected in its biological functions.

Proteins can be activated by internal(genes) or external (growth factor) stimuli. These stimuli generally cause theprotein to be modified by processes such as phosphorylation, acetylation or ubiquitination.Once a protein is activated it continues downstream to activate other proteinsin what’s called a signaling cascade. The ultimate protein activated in thiscascade is generally a protein that can translocate into the nucleus and bindto DNA. These DNA binding proteins control the cells functions by turning genesoff or on.  When it comes to cancer theprotein networks in the cell can become disturbed and may be up ordown-regulated to achieve immortality. This can be instigated at a genomic leveldue to overexpression, deletions and mutations in genes known as oncogenes ortumor suppressors. Ultimately the products of these genes are deregulatedproteins which will function in dynamic and complex signaling network.

Aberrantprotein functions can cause an entire disruption in cell signaling and lead todisturbed outcomes such as immortality. Focus has therefore shifted inidentifying and targeting these deregulated proteins (Pierotti et al., 2010). Deregulated proteins or the pathway they operate in, could then betargeted directly by anti-cancer therapeutics (Chae & Gonzalez-Angulo, 2014).  RPPA was first developed and utilized by(Paweletz et al., 2001). Thisstudy showed the effective use of RPPA in determining activated proteins(phosphorylated proteins) that may be involved with the development of prostatecancer.

Using RPPA they successfully captured a pro-survival protein at differentstages of prostate cancer (Reymond Sutandy et al., 2013). The PI3k/Akt pathway, previously implicated in cancerproliferation (Fresno Vara et al.

, 2004),involves the activation of downstream proteins by successive phosphorylation.Akt is one such downstream protein in which RPPA was used to demonstrate theincreased phosphorylation states of Akt in prostate cancer cells. Proteinlevels of Akt remained constant across samples, but phosphorylation increased. Another prostate cancer study also usedRPPA to show the changing states of phosphoproteins with disease progression (Grubb et al.

, 2003). It supported the result findings from above that AKTphosphorylation levels increased from normal cells transitioning to tumorigenic.Similarly phosphor-ERK levels showed a decrease from normal to tumor tissues. Before RPPA there was a gap in themarket for high throughput analysis of proteins that play key roles in disease.One such example is a group of primary immune disorders (PIDs) which are generallyleft undiagnosed. In some instances, where left untreated, the results can befatal.

C3 is an important protein that plays an essential role in thecomplement immune system. The study carried out by (Janzi et al., 2009) utilized dried blood spot samples (DBSS) from newborns and screenedthe samples, using RPPA, to test for the presence of C3. The results showedclear presence or absence of C3 in healthy and C3 deficient patients respectively.The DBSS were collected from newborns but were stored for up to 21 years beforeanalysis. Fresh samples were used as a control to ensure degradation of the C3protein did not occur over the 21 years. This highlights the unique advantageof fixing the proteins onto the surface.

By reverse phase the samples arepreserved on the glass side and can be stored for reuse. RPPA has the advantageous ability toscan a number of different samples for the same protein. Thus the proteinlevels from different subgroups can be compared and interpreted. This is illustratedconcisely in a paper published by (He et al.

, 2012). Here they compare the protein ku80 (a protein involved in DNAdamage repair) levels in tumors belonging to smokers’ vs non-smokers. Without surprise,the activated level of ku8 was far higher in smokers’ vs non-smokers,indicating DNA damage repair is occurring in smokers. Differences in proteinsinvolved in survival, proliferation and migration were also seen when normal vstumor samples were compared. These results were confirmed by western blot analysisand deemed statistically significant.  Breastcancer (BC) is one of the most prominent diseases affecting women in thismodern era. Genomic profiling offered a breakthrough in BC by characterizingthe complex disease into subtypes based on their receptor status (Perou et al.,2000).

The three main groups areLuminal (A and B), HER2 enriched and Triple Negative (TNBC). Luminal type BC haveboth estrogen (ER) and progesterone (PR) receptors and may also have the HER2receptor (luminal B). TNBC, as the name suggests, does not have the ER, PR or theHER2 receptor. It lends the lowest survival rates. In 2016, (Marcotte etal., 2016) undertook one of the largeststudies to classify over 82 BC cell lines into molecular subtypes. The studyalso grouped cell lines together on the basis of causal variants and potential vulnerabilities.RPPA was used in this study.

Cell lines expressing similar proteins, both phosphoand basal levels, were grouped together in clusters. Through the results of (Marcotte etal., 2016) BC cell lines which have beengrouped together on the basis of deregulated proteins, can be examined furtherwith the potential of classifying certain molecular subtypes on the basis oftheir deregulated or distinct proteome. Following this, a protein inhibitorcould then be developed that would perhaps successfully target these subtypes. Thisleads to the next and finalized thought in this review- personalized medicine. The future of medicine is changing. The needfor personalized medicine, particularly when it comes to cancer, is nowapparent.

RPPA is aiding that change and its new found role, aside from basicresearch, in clinical trials is emerging (Gallagher & Espina, 2014). In complex diseases such as cancer, the protein target couldpotentially differ from patient to another. This is seen in a number of caseswhereby therapeutics that are aimed at a particular cancer subtype do not shownear 100% success.

This is possibly accounted for the fact that individualsproteomes differ from patient to patient. Although they may be suffering fromthe same disease it may be due to different proteins involved in the same ordifferent pathways. Thus, the future of cancer research seems to be heading inone way- personalized therapeutics. A patients’ tissue sample could be subjectedto an RPPA to examine prominent acting proteins. These protein levels could becompared to that of other cancers of the same molecular subtype. Once apossible protein target has been identified RPPA could then be utilized to testthe efficiency or expected outcome of this drug. A number of ongoing clinical trialsexist whereby RPPA is being used to test a potential therapeutic agent (Mueller, Liotta, & Espina, 2010).

The ‘I-SPY’  (investigation ofserial studies to predict your therapeutically response with imaging andmolecular analysis) trials are a major group clinical trials currently takingplace in order to examine the potential effectiveness of a potential drug on aspecific individual. It is specific for breast cancer patients, wherebymethodologies, such as RPPA are being used to identify protein targets that areunique to that individual (Barker et al., 2009).  ConclusionsOverall, the reverse phase protein arrayhas a number of advantages which certainly outweigh its disadvantages.

It is arobust, sensitive and cost effective assay that supersedes the forward phasearrays. Aside from its use in basic research, thefuture of RPPA from a clinical standpoint seems obvious. After exhaustinggenomic analysis, tailored therapeutics directed at deregulated proteins couldprove a successful way in targeting complex diseases such as cancer. Beforethis is achieved antibody sensitivity and validation processes must bedeveloped so that deregulated proteins, even at low levels can be successfullydetected and targeted.


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