after decades of drawbacks , cancer immunotherapy finally started to deliver its promise . as far as radiation and conventional cytostatic drugs mainly achieved its pinnacle in therapeutic efficiency and
there is not much space left for their improvement , oncologists see the future of the anticancer treatment mainly in two approaches : targeted therapy and immunotherapy .
however , for most common human cancers such as breast and prostate cancers there were no specific targets found so far ( except her2 for a fraction of breast cancers ) .
an emerging approach to treat such tumors is to boost tumor - specific immune response .
this approach is based on a valid assumption that tumor development and growth is accompanied by suppression of host 's immune system and/or escaping from immune surveillance ; therefore stimulation of immunity or targeting hidden tumor antigens should lead to anti - tumor response .
gurin ) vaccine used to treat nonmuscle invasive form of bladder cancer is early successful example of such immune booster , which works , apparently , via stimulation of local innate immune response .
currently , there are also several immunotherapeutics approved by fda or in phase iii clinical trials , such as monoclonal antibodies to pd-1 and ctla-4 which are effective in metastatic melanoma .
their activity is based on relieving immunosuppression caused by tumors , and their efficiency in clinics ( although only in fraction of patients ) with rather mild toxicity is a proof of principle that immune system of a cancer patient is not completely disabled and can be awakened .
another mainstream of cancer immunotherapy is based on boosting patient 's immune response to cancer - specific antigens ( ag ) by vaccines .
accordingly , first were approved the vaccines against cervical cancer target ag of human papilloma viruses ( e.g. , e6/e7 antigens of hpv16 and hpv18 ) . except for head and neck cancer where hpv
is also involved , or hepatoma , where hcv plays a pathological role , a vast majority of human tumors , however , are not caused by viruses , therefore the choice of a tumor ag is challenging . furthermore , anticancer vaccines tested so far are not effective , in particular , since they apply selective pressure on cancer cells , which led to the loss of the vaccine - encoded ag ( immunoediting ) and resulted in the tumor relapse . as a result , despite numerous attempts , there is only one anticancer vaccine approved so far for common cancer ( provenge for prostate cancer ) , with pap ( prostate alkaline phosphatase ) as an ag .
however , this vaccine has rather limited effect ( increase in survival by only 4 month ) , requires sophisticated in vitro production , and is very expensive . despite these drawbacks
, it demonstrates that host 's immune system can be enforced to fight cancer , although with an additional help from in vitro propagated immune cells .
dna vaccine is an antigen - encoding vector which is administered to the patient in order to elicit immune response .
typically , a dna vaccine vector is a plasmid , circular double stranded bacterial dna .
dna vaccines enter myocytes and tissue - resident apc ( ag - presenting cells , e.g. , macrophages ) which results in intracellular synthesis of vaccine - encoded tumor antigen .
after the vaccine - encoded protein is expressed and processed , antigen - derived peptides are presented to nave t - cells with subsequent generation of ag - specific cytotoxic cd8 + t - cells ( ctl ) and humoral immune response [ 7 , 8 ] .
the key difference between a dna vaccine and a protein vaccine is that the antigen is expressed intracellularly . as a result ,
the antigen undergoes posttranslational modifications and antigen presentation through an entire array of naturally occurring intracellular mechanisms , which leads to several advantages . for example
, dna vaccines can induce very strong t - cell and b - cell responses even if amounts of antigen produced in situ is minimal .
for example , a dna vaccine can encode two forms of an antigen , proteosome - resistant and proteosome degradable forms .
it was shown that combination of these two forms elicits stronger immune response than either of these two forms alone . in the future ,
vaccine - encoded ag can be modified , so it can be produced in a form with an optimal rate of proteosome degradation of an encoded protein .
furthermore , an order to modulate an intracellular ag fate , dna vaccines can be engineered to express a tumor ag fused with an adjuvant protein , for example , polyglutamine sequence , inducing intracellular self - binding and aggregate formation of the vaccine - encoded antigen , which also lead to better immunogenicity . until recently , there were two significant drawbacks of dna vaccine methodology .
first , dna vaccines could not be used for cytotoxic proteins because high level of their expression would kill the vaccine - transfected cells .
second , for some ags , extracellular expression of their wild - type protein forms could lead to manifestation undesirable / toxic activity of the ag protein . at the same time
, it was not possible to inactivate negative functions of the ag protein introducing mutations into the plasmid , because a mutated gene possessed a very low expression level .
due to developments of past decade , a vaccine - encoded protein can be modified to eliminate its negative and/or dangerous properties while preserving all immunogenic domains . utilizing plasmids as a backbone provides significant benefits .
bacterial sequences such as unmethylated cpg islands in the plasmid vector operate as an adjuvant , stimulating activation of tlr9 . from the point of view of public health feasibility
, dna vaccines can be generated in large amounts and with clinical grade purity in inexpensive and rapid fashion ; they are safe and highly stable comparing to protein vaccines . despite their great promise dna vaccines face two major stumbling blocks : ( i ) immune response they elicit may be strong enough for small animals , but not for humans ( see ref .   for review ) ; and ( ii ) mutations introduced into the antigen may alter its mrna structure , which may lead to severe reduction in expression level .
nevertheless , there are several dna vaccines approved already for veterinary applications , including anti - melanoma dna vaccine ( oncept ) for dogs . among approaches to increase efficiency of anticancer dna vaccine in humans
are improving its administration ( e.g. , electroporation and gene gun which increases delivery of plasmid dna by several times ) and use of enhancers of immune response ( e.g. , gm - csf , il-2 ) , which can be also coded by plasmid dna .
use of viral vectors such as retroviruses , lentiviruses , and adeno ( or adeno - associated ) viruses , although increasing delivery of dna , but also have its limitation : possible carcinogenesis due to insertion of viruses in host genome , immunogenicity , broad tropism , limited packaging capacity , and difficulty of viral production . to avoid these drawbacks of viral vectors , current development of material sciences and nanotechnology , as well as in nucleic acid chemistry led to emergence of more efficient nonviral delivery system ( e.g. , lipid - based and polymeric ) some of which are now being tested in clinical trials ( see ref .   for review ) .
according to clinicaltrials.gov , there are 190 trials listed of cancer treatment which used or currently using dna vaccination alone or with some combination . among targeted cancer ag are e6/e7 proteins of hpv-16 for cervical cancer , alpha - fetoprotein for liver cancer , cea for colon cancer , trp2 for melanoma , pap for prostate cancer and some others .
furthermore , there are several dna vaccines approved already for veterinary applications , including antimelanoma dna vaccine ( oncept ) for dogs , demonstrating feasibility of dna vaccination .
in 2009 , a group of specialists in cancer immunotherapy made an attempt to prioritize cancer antigens for acceleration of translational research ( national cancer institute pilot project ) .
they created nine criteria and evaluated 75 tumor antigens being studied in clinics and preclinically at that time .
the first ranking criterion ( weighting 31% ) , unsurprisingly , was therapeutic function ( i.e. , at least some efficiency in clinical trials ) , then , in descending order : ( 2 ) immunogenicity ; ( 3 ) role of agin oncogenicity ; ( 4 ) specificity ; ( 5 ) expression levels and percent of antigen - positive cancers ; ( 6 ) stem cell expression ; ( 7 ) number of patients with ag - positive cancers ; ( 8) number of antigenic epitopes ; ( 9 ) cellular location of ag expression ; first four criteria were the most important , giving in total 79% contribution in prioritization . if we exclude first therapeutic criterion to choose an ag for preclinical studies , there will be only three major criteria left .
in other words , ideally , an antigen for anticancer vaccine should be : ( i ) immunogenic ; ( ii ) essential for cancers cells ( to avoid its loss through immune - editing ) , but dispensable for normal tissues to reduce the risk of toxicity , and ( iii ) overexpressed in tumors as compared to the normal tissues . we hypothesized that p62 protein ( sequestome 1 ) may be such an excellent target as a cancerag .
structure of p62 ( sqsm1 ) and functions of its domains in cell signaling and protein degradation .
p62 protein is a major player in selective macroautophagy   and serves as a signaling hub for several signal transduction pathways , among them nf - kb , traf6 , map kinases , twist1 etc .
[ 1922 ] ( figure 1 ) importantly , p62 is dispensable for normal tissues , but essential for development and survival of tumors ( table 1 ) .
first , p62 knockout mice are viable and demonstrate only minor anomalies ( later - onset obesity ) , indicating that , at least under normal conditions , other proteins can substitute functions of p62 ( e.g. , p62 homolog nbr1 in autophagy ) . but as for tumor development , the situation is quite different .
at least in several mouse models studied , knockout of p62 prevented or markedly delayed development of cancer caused by oncogenes ( table 1 ) . furthermore , fully transformed cells do not lose its dependence on p62 since its knockdown causes inhibition of growth or loss of viability ( table 1 ) .
although p62 is not oncogene per se , such dependence of tumors on some proteins is a well - known phenomenon called  nononcogene  addiction . according to this notion , proteins dispensable for normal cells become necessary for transformed cells . along with oncogenes such as myc and ras whose inactivation leads to tumor suppression or even eradication , these nononcogenic proteins are also considered as good therapeutic targets especially when oncogenes are difficult to find or they are undruggable . which of the p62 activities are critical for tumorigenesis ?
it appears that autophagy at early stages of tumor development is not required but rather detrimental . at these stages , p62 is important as a signaling hub , in particular , by activation of proinflammatory and prosurvival nf - kb pathway [ 19 , 20 , 26 , 27 ] or twist1 , which is involved in cell proliferation , migration , and metastases   ( figure 1 ) .
in contrast , at later stages of tumor development , autophagy plays a distinct protective role , and its inhibition per se has antitumor effect .
some tumors such as pancreatic cancer become addicted to autophagy , and accordingly , there are several ongoing clinical trials with autophagy inhibitor chloroquine alone or in combination with chemotherapy [ 28 , 29 ] . whatever the mechanisms of tumor dependence on p62 , this is apparently a wide - spread phenomenon ( table 1 ) .
another requirement for being a good agfor anticancer vaccine is the higher expression level in tumor tissue . indeed , according to data of oncomine ( the largest database of human cancer microarrays ) and other reports , at least 10 various types of human cancer have high levels of p62 comparing to normal tissue ( table 1 ) .
for instance , in melanoma , p62 levels are 10-times higher ( oncomine ) . furthermore , in several types of tumors , levels of p62 increase during progression ( table 1 )
we have found that common oncogenes such ras , pik3ca , and her2 can induce p62 in vitro . in addition , tumor microenvironment such as inflammation and oxidative stress can activate p62 via nf - kb   and nrf-2   transcription factors .
interestingly , activation of these transcription factors also depends on p62 which leads to a positive feed - back loop [ 27 , 31 ] ( figure 1 ) .
the majority of cancer ags targeted so far are extracellular membrane proteins , but p62 is an intracellular protein .
however , recently accumulated data indicate that intracellular proteins also can be feasible ags for antibody response , for instance survivin , prl-3 cancer - associated phosphatase , [ 3234 ] , or wt1 .
there are several proposed mechanisms of targeting of these proteins by immune system : ( i ) uptake of antibodies ( e.g. , via endocytosis ) and neutralization of the ag ; and ( ii ) displaying of the ag on the surface via unconventional secretion pathway ; [ 36 , 37 ] . relative contribution of these mechanisms in immune response to intracellular agis currently unknown , but at least in case of prl-3 oncoprotein , both b - cells and nk cells seems to be necessary , whereas t - cells are dispensable . at the same time , antibody to wt1 was effective even in scid mice ( i.e. , without both t- and b - cells ) .
last but not least , proteolytic fragments of intracellular p62 may be presented by mhc - i molecules to attract ctl , which makes dna vaccines and dna vaccination approach particularly attractive .
based on above considerations , we have chosen p62 was as an ag for a dna vaccine and evaluated its antitumor effect . in studies of hundreds of animals ,
p62 vaccine has proven its effectiveness in five kinds of solid tumors in mice and rats : lung and breast carcinomas , melanoma and sarcoma ( table 2 ) .
more importantly , it also possessed strong antimetastatic activity in three models of metastases : spontaneous metastases to lung ( lewis lung carcinoma ) , to regionary lymph nodes ( sarcoma 37 ) , and induced metastases ( by i.v .
we also found that , at least in case of lung carcinoma and melanoma , p62 vaccine decreased both the number and size of metastasis , indicating that it suppresses both colonization of lung by tumor cells ( e.g. , formation of micrometastases ) , as well as growth of established metastases . of note , in case of melanoma , p62 vaccine was effective not only in preventive ( given 2 weeks before tumor inoculation ) , but in therapeutic setting as well ( after tumor inoculation ) , suppressing both primary tumor and metastases .
this metastatic melanoma model , where tumor cells are already in circulation ( after i.v .
injection ) and p62 being injected afterwards more closely resembles human cancer , where many patients are first diagnosed with cancer when metastatic process is already underway . since great majority of patients ( about 90% ) die from metastases rather than from primary tumors , such antimetastatic effect of p62 vaccine seems very encouraging . also encouraging
are our preliminary data about the effect of the vaccine on spontaneous tumors in dogs , in particular , mammary tumors . given on compassionate use basis to several dogs with incurable cancers , p62 vaccine halted the progression of the disease and
markedly improved animal 's well - being ( manuscript in preparation ) . as a next step in preclinical development of p62 dna vaccine as a candidate for future clinical trials
, its toxicological properties were tested in mice , rats , guinea pigs , and dogs .
as one can see from table 3 , the vaccine was well - tolerated , without acute or chronic toxicity , allergic activity , and it did not cause embryonic toxicity and teratogenicity . there was no hematotoxic , hepatotoxic , and nephrotoxic effects , as well as effects on carbohydrate and lipid metabolism .
furthermore , histological examination of brain , heart , lung , liver , kidney , spleen , thyroid , and thymus did not find any anomalies ( shifrin et  al .
. therefore , antitumor and anti - metastatic activity of p62 vaccine was not accompanied by any significant side effects , as expected for dna vaccines .
overall , preclinical data presented above justifies further veterinary research and human clinical testing of p62-encoding dna vaccine as a novel anticancer agent .
the authors are employees of curelab oncology inc . the authors alone are responsible for the content and writing of the article .
 cancer immunotherapy is a thriving field , but its clinical achievements are modest so far . 
 one of its major hurdles seems to be finding a feasible cancer antigen as a target for immune response . 
 after many years of research , three major criteria for choice of tumor antigens emerged . 
 an antigen should be : ( i ) immunogenic ; ( ii ) essential for cancers cells ( to avoid its loss through immunoediting ) , but dispensable for normal tissues to reduce the risk of toxicity , and ( iii ) overexpressed in tumors as compared to the normal tissues . 
 here we argue that p62 ( sqstm1 ) , a protein involved in autophagy and signal transduction , fits all the above criteria and can be chosen as a novel cancer antigen . 
 accordingly , we carried out an extensive study and found antitumor and antimetastatic activity of p62-encoding dna vaccine in five types of commonly used transplantable tumor models of mice and rats , and spontaneous tumors in several dogs . given that toxicity of p62 vaccine was minimal , if any , we believe that p62-encoding vaccine merits further clinical development . 
INTRODUCTION
DNA VACCINES
P62 AS A NEW CANCER ANTIGEN
ANTITUMOR EFFECT OF P62 DNA VACCINE
FUNDING
Declaration of Interests