presently in the united states , it is estimated that one in five people will develop some form of skin cancer due to a combination of family history , environment , and genetic factors ( see the american cancer society for more information , http://www.cancer.org/cancer/skincancer-melanoma/detailed guide / melanoma - skin - cancer - risk - factors ) .
dna damage caused by ultraviolet irradiation is thought to be the main environmental culprit behind the development of this disease .
likewise , genetic disruption of genes necessary for the appropriate balances between survival , proliferation , and cell death are frequently associated with the development and persistence of cells that result in skin cancer .
these cancerous cells may also develop mechanisms to evade the immune system resulting in unimpeded cell proliferation , survival , and metastases .
malignant melanoma is the deadliest form of skin cancer , and it arises from the uncontrollable cell division of the basal layer melanin - producing cells called melanocytes ( for in - depth review , see ibrahim and haluska , 2009 ) .
melanoma commonly involves the skin , but it can occur at other sites such as the eyes , ears , and gastrointestinal tract . while melanoma only accounts for 5% of skin cancers , it is responsible for the majority of skin cancer deaths ( jemal et al . , 2009 ) .
the prognosis of melanoma is favorable when limited to the skin and wide excision is curative .
however , the prognosis of metastatic melanoma is very poor with a 5-year survival rate of less than 20% . presently , the most used anti - tumor strategy for stage iv metastatic melanoma is dacarbazine ( ditc ) , which as a single treatment is not very effective , and is associated with < 20% response rate ( tsao et al . , 2004 ) .
adjuvant and combination treatments are also used ; for example , cisplatin , vinblastine , and dtic ( the regimen is referred to as  cvd  ) , in combination with interferon alpha-2 ( ifn ) and interleukin-2 ( il-2 ) , but none have proven to be significantly effective , yet they demonstrate substantial toxicity ( legha et al . , 1989 , 1996 ; phan et al . , 2001 ) .
in the majority of cases where a response does occur , there is often recurrence of the cancer within months
. a better understanding of the pathways that are activated to promote melanoma , and targeted during treatment , will hopefully reveal novel therapeutic opportunities . toward the end of our discussion
, we will propose that manipulating the bcl-2 family and the mitochondrial pathway may offer a potential therapeutic inroad to treatment success . in response to chemotherapeutics , cells attempt to eliminate the damage or recover from it by engaging stress response pathways ( e.g. , the p53 pathway ) , but often the damage is overwhelming , and the treated cells induce a form of programmed cell death known as apoptosis ( green and evan , 2002 ) .
apoptotic signaling can initiate from outside the cell via plasma membrane receptors ( referred to as the  extrinsic pathway  ; e.g. , cd95/fas or tumor necrosis factor receptor , tnfr ) , or through stress that originates from within the cell ( referred to as the  intrinsic  or  mitochondrial pathway  ; e.g. , macromolecular damage ; guicciardi and gores , 2009 ; chipuk et al . ,
the extrinsic apoptotic pathway is engaged when pro - apoptotic ligands such as tnf or cd95l / fasl trigger death receptor signaling which directly leads to the activation of caspases , which are the cysteine 
aspartic proteases responsible for inducing the apoptotic phenotype ( e.g. , dna laddering , loss of plasma membrane asymmetry , and cellular blebbing ; pop and salvesen , 2009 ) .
the intrinsic pathway is triggered by intracellular stress signals such as dna damage , oncogenes , hypoxia , and growth factor withdrawal ( chipuk et al . ,
these cellular stressors transcriptionally and post - transcriptionally regulate the b cell cll / lymphoma-2 ( bcl-2 ) family of proteins , which are responsible for mitochondrial integrity , subsequent caspase activation , and apoptosis .
while these two general pathways are distinct , there are situations of cross - talk where the extrinsic pathway promotes apoptosis through the intrinsic pathway and vice versa . in these situations ,
the bcl-2 family of proteins is also crucial control points of cellular fate . for the most part ,
cancer cells utilize and respond to chemotherapeutic agents by regulating the mitochondrial pathway of apoptosis , so our discussions will be focused on these signaling mechanisms ( figure 1 ) . the mitochondrial pathway of apoptosis . following cellular stress such as dna damage ,
the bcl-2 family of proteins is regulated by transcriptional ( increased or decreased ) and post - translational ( e.g. , phosphorylation , cleavage , and relocalization ) mechanisms .
if the cellular stress is irreparable , the culmination of pro - apoptotic signaling will lead to mitochondrial outer membrane permeabilization ( momp ) , which allows for the release of soluble intermembrane space proteins , including cytochrome c. apaf-1 , cytochrome c , and datp then coordinate to promote apaf-1 oligomerization and recruitment of pro - caspase-9 ; this complex is referred to as the  apoptosome . 
the apoptosome allows for pro - caspase-9 dimerization and activation , and the subsequent cleavage and activation of executioner caspases ( caspase-3 , -6 , and -7 ) .
these caspases promote the characteristic phenotypes of apoptosis , e.g. , chromatin condensation , dna laddering , and plasma membrane blebbing , by cleaving numerous intracellular substrates . within the body ,
apoptotic cells are rapidly cleared by phagocytosis to ensure tissue homeostasis . once the apoptotic program has been initiated by compromising the outer mitochondrial membrane ( omm ) ,
pro - apoptotic factors are released from the mitochondrial intermembrane space ( ims ) , including cytochrome c ( figure 1 ; liu et al . , 1996 ) .
monomeric apoptotic protease activating factor 1 ( apaf-1 ) cooperates with cytochrome c and datp to form the oligomeric caspase activation platform referred to as the  apoptosome  ( li et al . , 1997 ;
the apoptosome recruits , dimerizes , and activates pro - caspase-9 , which then promotes the downstream function of pro - caspases-3 , -6 , and -7 ( figure 1 ) . often , this step in the mitochondrial pathway is referred to as  the point of no return ,  as mitochondrial function and atp generation gradually wane , and proteolytic cleavage of caspase substrates ensues .
caspase activity is responsible for the morphological phenotypes of apoptosis , including dna laddering , loss of plasma membrane asymmetry , and phagocytosis ( figure 1 ; logue and martin , 2008 ) .
regulation of the mitochondrial pathway of apoptosis plays a crucial role in the development and treatment of melanoma ; and it has been shown that defects in this pathway can result in resistance to tumor suppressor pathways and treatments ( for an in - depth review , see soengas and lowe , 2003 ) .
the involvement of mitochondria in apoptosis was first suggested when caspase activity resulted from xenopus oocyte extract co - incubation with purified mitochondria ( newmeyer et al . , 1994 ) .
this activity was blocked by the addition of bcl-2 , suggesting that bcl-2 could prevent mitochondrial engagement of the cytosol . since then
, the bcl-2 family has grown to include almost 20 members that are divided into two functional classes of proteins : anti - apoptotic and pro - apoptotic ( figure 2 ) .
most cells express a variety of anti - apoptotic and pro - apoptotic bcl-2 proteins , and through the regulation of their interactions command survival or commitment to apoptosis ( for an in - depth review , see chipuk and green , 2008 ) .
the bcl-2 family of proteins is divided into anti - apoptotic and pro - apoptotic members .
the anti - apoptotic members include : a1 , bcl-2 , bcl - w , bcl - xl , and mcl-1 , all of which contain four bcl-2 homology domains ( termed bh1 - 4 ) .
the pro - apoptotic proteins are subdivided into  effector  and  bh3-only  members .
the effector proteins , bak and bax , also contain bh1 - 4 ; while the bh3-only proteins contain only one domain , a bh3 , that is required for interactions with anti - apoptotic and effector proteins .
the bh3-only proteins include : bad , bid , bik , bim , bmf , bnip3 , hrk , noxa , and puma .
bcl-2 core  structural unit , which includes a hydrophobic groove that binds to bh3 domains .
anti - apoptotic bcl-2 proteins are comprised of four bcl-2 homology domains ( bh1 - 4 ) and are generally integrated within the omm , but may be present in other membranes like the endoplasmic reticulum ( petros et al . , 2004 ) .
bcl-2 , bcl - xl , and myeloid cell leukemia 1 ( mcl-1 ) are the major members of the anti - apoptotic bcl-2 repertoire that function to preserve omm integrity by directly binding and inhibiting the pro - apoptotic bcl-2 proteins ( chen et al . , 2005 ; willis et al .
the pro - apoptotic bcl-2 members are divided into effectors ( which also contain bh1 - 4 ) and the bh3-only proteins ( figure 2 ) .
the effector proteins bak ( bcl-2 antagonist killer 1 ) and bax ( bcl-2 associated x protein ) homo - oligomerize into proteolipid pores within the omm and are required to promote momp and cytochrome c release ( lindsten et al . , 2000 ; wei et al . , 2001 ) .
however , these effectors require an activation step , upon which they oligomerize and gain the capacity to permeabilize membranes composed of mitochondrial lipids ( kuwana et al . , 2002 ) .
activation of bak and bax occurs through interaction with so - called  direct activators  ( see below ) , or by physico - chemical effects of detergents , mild heat , or elevated ph ( hsu and youle , 1997 ; khaled et al . , 2001 ; letai et al . , 2002 ; pagliari et al . ,
the bh3-only proteins act to regulate both the anti - apoptotics and effectors ( figures 2 and 3 ) .
two of these , bid ( bh3 interacting domain death agonist ) and bim ( bcl-2 interacting mediator of cell death ) , are direct activators of bak and bax , acting via their bh3 domains to induce oligomerization and the permeabilization function of bak and bax ( wei et al . , 2000 ; kuwana et al . , 2002 , 2005 ; letai et al . ,
the process by which bak and bax permeabilize the omm is commonly referred to as  mitochondrial outer membrane permeabilization ,  or momp . mechanisms of action within the bcl-2 family leading to momp .
the upstream involvement of the bcl-2 family in apoptosis can be divided into two parallel activities : de - repression and sensitization .
the de - repression model begins at cellular status quo with anti - apoptotic bcl-2 proteins binding and sequestering bh3-only direct activators ( e.g. , bcl-2 binds bim ) . in response to stress , a de - repressor bh3-only protein is induced ( e.g. , bad ) , which can then displace the direct activator and liberate it to activate bak and bax .
the sensitization model involves the preemptive binding and inactivation of anti - apoptotic proteins by sensitizer bh3-only proteins ( e.g. , bcl-2 binds bad ) .
this sensitization prevents the inhibition of direct activator bh3-only proteins induced following apoptotic stimulation ( e.g. , bim ) , thus lowering the threshold level of direct activator necessary to activate the effectors bak and bax .
unbound direct activator bh3-only proteins function in the direct activation of bak and bax through a transient interaction .
these interactions lead to conformational changes involving n - terminal rearrangement , which exposes the bh3 domain of bak and bax .
activated bak monomers pair symmetrically , and oligomerize as sets of dimers to permeabilize the outer mitochondrial membrane leading to momp .
recent in vitro studies show that activated bid and bax bind to one another only in association with membranes , and this precedes bax oligomerization , which is followed by membrane permeabilization ( lovell et al . , 2008 ) .
the accepted viewpoint is that the expression , stability , and activation of the bh3-only proteins and their interactions with the anti - apoptotic and effector proteins sufficiently links pro - apoptotic signal transduction to momp(chipuk et al . , 2010 ) . broadly speaking ,
regulation of the bh3-only proteins at the transcriptional and translational levels determines the omm integrity .
when an apoptotic signal is initiated , the anti - apoptotic bcl-2 proteins are inhibited by the sensitizer / de - repressor bh3-only proteins ( e.g. , bad , bmf , hrk , noxa , and puma ) , which allows for direct activator - induced bak and bax activation to promote momp , and subsequent apoptosis ( figure 3 ) .
non - bcl-2 family direct activator proteins are also described ; several reports suggest a direct activator function for cytosolic p53 , map-1 , and asc ( tan et al . , 2001 ;
furthermore , controversy exists regarding the role of puma ( p53 upregulated modulator of apoptosis ) in mediating bak and bax activation , as conflicting results suggest that puma is either a de - repressor / sensitizer or direct activator bh3-only protein ( chipuk et al . , 2005 ; kuwana et al . ,
finally , the steps leading to bak and bax activation have been unveiled through studies that focused on structural consequences of bim and bak / bax interactions ; these structural details are discussed in figure 3 ( dewson et al .
in terms of chemotherapeutic success , pushing a cell to undergo momp is suggested to enhance clinical outcomes , and this is highlighted by the development of small molecule inhibitors to the anti - apoptotic bcl-2 proteins , which lower the cellular threshold for momp and apoptosis ( letai , 2008 ) .
in response to chemotherapeutics , cells attempt to eliminate the damage or recover from it by engaging stress response pathways ( e.g. , the p53 pathway ) , but often the damage is overwhelming , and the treated cells induce a form of programmed cell death known as apoptosis ( green and evan , 2002 ) .
apoptotic signaling can initiate from outside the cell via plasma membrane receptors ( referred to as the  extrinsic pathway  ; e.g. , cd95/fas or tumor necrosis factor receptor , tnfr ) , or through stress that originates from within the cell ( referred to as the  intrinsic  or  mitochondrial pathway  ; e.g. , macromolecular damage ; guicciardi and gores , 2009 ; chipuk et al .
the extrinsic apoptotic pathway is engaged when pro - apoptotic ligands such as tnf or cd95l / fasl trigger death receptor signaling which directly leads to the activation of caspases , which are the cysteine 
aspartic proteases responsible for inducing the apoptotic phenotype ( e.g. , dna laddering , loss of plasma membrane asymmetry , and cellular blebbing ; pop and salvesen , 2009 ) .
the intrinsic pathway is triggered by intracellular stress signals such as dna damage , oncogenes , hypoxia , and growth factor withdrawal ( chipuk et al . , 2010 ) .
these cellular stressors transcriptionally and post - transcriptionally regulate the b cell cll / lymphoma-2 ( bcl-2 ) family of proteins , which are responsible for mitochondrial integrity , subsequent caspase activation , and apoptosis . while these two general pathways are distinct , there are situations of cross - talk where the extrinsic pathway promotes apoptosis through the intrinsic pathway and vice versa . in these situations ,
the bcl-2 family of proteins is also crucial control points of cellular fate . for the most part ,
cancer cells utilize and respond to chemotherapeutic agents by regulating the mitochondrial pathway of apoptosis , so our discussions will be focused on these signaling mechanisms ( figure 1 ) . the mitochondrial pathway of apoptosis . following cellular stress such as dna damage ,
the bcl-2 family of proteins is regulated by transcriptional ( increased or decreased ) and post - translational ( e.g. , phosphorylation , cleavage , and relocalization ) mechanisms .
if the cellular stress is irreparable , the culmination of pro - apoptotic signaling will lead to mitochondrial outer membrane permeabilization ( momp ) , which allows for the release of soluble intermembrane space proteins , including cytochrome c. apaf-1 , cytochrome c , and datp then coordinate to promote apaf-1 oligomerization and recruitment of pro - caspase-9 ; this complex is referred to as the  apoptosome . 
the apoptosome allows for pro - caspase-9 dimerization and activation , and the subsequent cleavage and activation of executioner caspases ( caspase-3 , -6 , and -7 ) .
these caspases promote the characteristic phenotypes of apoptosis , e.g. , chromatin condensation , dna laddering , and plasma membrane blebbing , by cleaving numerous intracellular substrates . within the body ,
once the apoptotic program has been initiated by compromising the outer mitochondrial membrane ( omm ) , pro - apoptotic factors are released from the mitochondrial intermembrane space ( ims ) , including cytochrome c ( figure 1 ; liu et al . , 1996 ) .
monomeric apoptotic protease activating factor 1 ( apaf-1 ) cooperates with cytochrome c and datp to form the oligomeric caspase activation platform referred to as the  apoptosome  ( li et al . , 1997 ; zou et al . ,
the apoptosome recruits , dimerizes , and activates pro - caspase-9 , which then promotes the downstream function of pro - caspases-3 , -6 , and -7 ( figure 1 ) . often , this step in the mitochondrial pathway is referred to as  the point of no return ,  as mitochondrial function and atp generation gradually wane , and proteolytic cleavage of caspase substrates ensues .
caspase activity is responsible for the morphological phenotypes of apoptosis , including dna laddering , loss of plasma membrane asymmetry , and phagocytosis ( figure 1 ; logue and martin , 2008 ) .
regulation of the mitochondrial pathway of apoptosis plays a crucial role in the development and treatment of melanoma ; and it has been shown that defects in this pathway can result in resistance to tumor suppressor pathways and treatments ( for an in - depth review , see soengas and lowe , 2003 ) .
the involvement of mitochondria in apoptosis was first suggested when caspase activity resulted from xenopus oocyte extract co - incubation with purified mitochondria ( newmeyer et al . , 1994 ) .
this activity was blocked by the addition of bcl-2 , suggesting that bcl-2 could prevent mitochondrial engagement of the cytosol . since then
, the bcl-2 family has grown to include almost 20 members that are divided into two functional classes of proteins : anti - apoptotic and pro - apoptotic ( figure 2 ) .
most cells express a variety of anti - apoptotic and pro - apoptotic bcl-2 proteins , and through the regulation of their interactions command survival or commitment to apoptosis ( for an in - depth review , see chipuk and green , 2008 ) .
the bcl-2 family of proteins is divided into anti - apoptotic and pro - apoptotic members .
the anti - apoptotic members include : a1 , bcl-2 , bcl - w , bcl - xl , and mcl-1 , all of which contain four bcl-2 homology domains ( termed bh1 - 4 ) .
the pro - apoptotic proteins are subdivided into  effector  and  bh3-only  members .
the effector proteins , bak and bax , also contain bh1 - 4 ; while the bh3-only proteins contain only one domain , a bh3 , that is required for interactions with anti - apoptotic and effector proteins .
the bh3-only proteins include : bad , bid , bik , bim , bmf , bnip3 , hrk , noxa , and puma .
bcl-2 core  structural unit , which includes a hydrophobic groove that binds to bh3 domains .
anti - apoptotic bcl-2 proteins are comprised of four bcl-2 homology domains ( bh1 - 4 ) and are generally integrated within the omm , but may be present in other membranes like the endoplasmic reticulum ( petros et al . , 2004 ) .
bcl-2 , bcl - xl , and myeloid cell leukemia 1 ( mcl-1 ) are the major members of the anti - apoptotic bcl-2 repertoire that function to preserve omm integrity by directly binding and inhibiting the pro - apoptotic bcl-2 proteins ( chen et al . , 2005 ; willis et al . , 2005 , 2007 ;
the pro - apoptotic bcl-2 members are divided into effectors ( which also contain bh1 - 4 ) and the bh3-only proteins ( figure 2 ) .
the effector proteins bak ( bcl-2 antagonist killer 1 ) and bax ( bcl-2 associated x protein ) homo - oligomerize into proteolipid pores within the omm and are required to promote momp and cytochrome c release ( lindsten et al . , 2000 ;
however , these effectors require an activation step , upon which they oligomerize and gain the capacity to permeabilize membranes composed of mitochondrial lipids ( kuwana et al . , 2002 ) .
activation of bak and bax occurs through interaction with so - called  direct activators  ( see below ) , or by physico - chemical effects of detergents , mild heat , or elevated ph ( hsu and youle , 1997 ; khaled et al . , 2001 ; letai et al . , 2002 ;
the bh3-only proteins act to regulate both the anti - apoptotics and effectors ( figures 2 and 3 ) .
two of these , bid ( bh3 interacting domain death agonist ) and bim ( bcl-2 interacting mediator of cell death ) , are direct activators of bak and bax , acting via their bh3 domains to induce oligomerization and the permeabilization function of bak and bax ( wei et al . ,
2000 ; kuwana et al . , 2002 , 2005 ; letai et al . , 2002 ) .
the process by which bak and bax permeabilize the omm is commonly referred to as  mitochondrial outer membrane permeabilization ,  or momp . mechanisms of action within the bcl-2 family leading to momp .
the upstream involvement of the bcl-2 family in apoptosis can be divided into two parallel activities : de - repression and sensitization .
the de - repression model begins at cellular status quo with anti - apoptotic bcl-2 proteins binding and sequestering bh3-only direct activators ( e.g. , bcl-2 binds bim ) . in response to stress , a de - repressor bh3-only protein is induced ( e.g. , bad ) , which can then displace the direct activator and liberate it to activate bak and bax .
the sensitization model involves the preemptive binding and inactivation of anti - apoptotic proteins by sensitizer bh3-only proteins ( e.g. , bcl-2 binds bad ) .
this sensitization prevents the inhibition of direct activator bh3-only proteins induced following apoptotic stimulation ( e.g. , bim ) , thus lowering the threshold level of direct activator necessary to activate the effectors bak and bax .
unbound direct activator bh3-only proteins function in the direct activation of bak and bax through a transient interaction .
these interactions lead to conformational changes involving n - terminal rearrangement , which exposes the bh3 domain of bak and bax .
activated bak monomers pair symmetrically , and oligomerize as sets of dimers to permeabilize the outer mitochondrial membrane leading to momp .
recent in vitro studies show that activated bid and bax bind to one another only in association with membranes , and this precedes bax oligomerization , which is followed by membrane permeabilization ( lovell et al .
the accepted viewpoint is that the expression , stability , and activation of the bh3-only proteins and their interactions with the anti - apoptotic and effector proteins sufficiently links pro - apoptotic signal transduction to momp(chipuk et al . , 2010 ) .
broadly speaking , regulation of the bh3-only proteins at the transcriptional and translational levels determines the omm integrity . when an apoptotic signal is initiated , the anti - apoptotic bcl-2 proteins are inhibited by the sensitizer / de - repressor bh3-only proteins ( e.g. , bad , bmf , hrk , noxa , and puma ) , which allows for direct activator - induced bak and bax activation to promote momp , and subsequent apoptosis ( figure 3 ) .
non - bcl-2 family direct activator proteins are also described ; several reports suggest a direct activator function for cytosolic p53 , map-1 , and asc ( tan et al .
furthermore , controversy exists regarding the role of puma ( p53 upregulated modulator of apoptosis ) in mediating bak and bax activation , as conflicting results suggest that puma is either a de - repressor / sensitizer or direct activator bh3-only protein ( chipuk et al . , 2005 ; kuwana et al . , 2005 ; kim et al . ,
finally , the steps leading to bak and bax activation have been unveiled through studies that focused on structural consequences of bim and bak / bax interactions ; these structural details are discussed in figure 3 ( dewson et al . , 2008 , 2009 ;
in terms of chemotherapeutic success , pushing a cell to undergo momp is suggested to enhance clinical outcomes , and this is highlighted by the development of small molecule inhibitors to the anti - apoptotic bcl-2 proteins , which lower the cellular threshold for momp and apoptosis ( letai , 2008 ) .
melanoma is notorious for chemoresistance , and there continues to be tremendous research focused on identifying the molecular mechanisms to explain this phenotype ( for an in - depth review , see hocker et al . ,
unlike many other cancers , melanoma rarely displays mutations within the tumor suppressor p53 pathway , which does afford the opportunity to pharmacologically regulate the p53 pathway through chemotherapy - induced dna damage and stress - responses , and we will discuss this throughout ( hocker and tsao , 2007 ) .
however , mutations in n - ras [ neuroblastoma ras viral ( v - ras ) oncogene homolog ] and b - raf ( v - raf murine sarcoma viral oncogene homolog b1 ) are common events in melanoma , which are observed in approximately 20 and 70% of patients , respectively ( shukla et al . , 1989 ; davies et al . , 2002 ; reifenberger et al . , 2004 ; edlundh - rose et al . , 2006 ) .
additionally , the loss of pten ( phosphatase and tensin homolog ) expression , activation of the akt / pi3k ( phosphatidylinositol 3-kinases ) pathway , and loss of cdkn2a ( cyclin - dependent kinase inhibitor 2a ) often synergize with b - raf mutations leading to the progression from benign nevus , primary tumor , to metastatic disease ( haluska and hodi , 1998 ; kim , 2010 ; figure 4 ) .
melanoma originates from melanocytes in the skin that acquire a series of changes that promote the progression from a normal phenotype to a nevus , primary tumor , and eventual metastatic disease .
epigenetically silenced , mutated , or deleted genes that promote disease progression are listed and discussed throughout the text . in brief ,
common mutations observed early in melanoma development are in the genes encoding n - ras and b - raf , and these are often complemented by mutations in the melanocyte regulators such as mitf and bcl-2 .
subsequent changes to pten , cdkn2a , and mh2a along with activating mutations of the akt / pi3k pathway synergize with early mutations .
subsequently , alterations in pro - apoptotic sensitivity upstream ( e.g. , p53 ) and downstream ( e.g. , apaf-1 ) of the mitochondrial pathway of apoptosis are suggested . in parallel to these events
, the expression and function of the bcl-2 family is altered to establish marked resistance to pro - apoptotic stimulation : there is increased expression of anti - apoptotic members , and coordinated downregulation of pro - apoptotic members . activating mutations in n - ras and b - raf lead to positive influences on the cell cycle , mainly by promoting mitosis and preventing cells from engaging their repair and stress machineries , which leads to increased stress and accumulated mutations .
studies also suggest that the expression of akt / pi3k is directly correlated with melanoma progression ; and pten expression is subsequently lost by genetic and epigenetic mechanisms to further promote akt / pi3k survival pathways ( steck et al . , 1997 ;
indeed , a recent mouse model of combined b - raf v600e and pten loss provides in vivo evidence that these two pathways are necessary and sufficient to promote metastatic melanoma formation ( dankort et al . ,
cells can engage inhibitors to the cell cycle , for example cdkn2a , which encodes for the proteins p16 ( cyclin - dependent kinase inhibitor 2a ) and p14 ( alternative reading frame ) ; yet cdkn2a is also mutated , genetically lost , or epigenetically silenced in a significant percentage of melanomas ( castellano and parmiani , 1999 ) .
there are numerous other genes that are targeted during the transition between primary tumor and metastasis ; more recently , the histone variant macroh2a was shown to suppress tumor progression of malignant melanoma ( kapoor et al . , 2010 ) .
in brief , the sum of these mutations leads to an aggressive disease that is highly resistant to chemotherapeutics ( figure 4 ) . for this discussion
, we are focused on understanding how melanomas with significant alterations in n - ras , b - raf , and the other pathways listed above fail to engage apoptosis despite aberrations in cellular signaling leading to unregulated proliferation ( figure 4 ) .
more importantly , can we pharmacologically regulate these aberrant pathways to produce better responses in the clinic ? the answers to both of these questions point in the direction of the bcl-2 family of proteins . here , we suggest that this family of anti- and pro - apoptotic members may be what allows for melanoma development , survival , and chemoresistance , yet it offers a pharmacologically tractable achilles heel for successful combination / adjuvant therapies to treat malignant melanoma . in general , there are two approaches to block apoptosis that is normally engaged following deregulated cellular proliferation : the cell may decrease the expression of pro - apoptotic bcl-2 proteins ( e.g. , bax or bim ) ; or the cell can over - express anti - apoptotic bcl-2 proteins ( e.g. , bcl-2 or mcl-1 ) . in either case , the resulting phenotype is the failure to promote bak / bax activation , momp , and apoptosis following deregulated cellular proliferation cues or chemotherapeutic treatment .
this ultimately leads to metastatic disease with poor prognosis and decreased long - term survival despite chemotherapeutic intervention .
returning to the pathways that promote melanoma development and chemoresistance , it is widely regarded that b - raf v600e is the most commonly observed mutation in patients ( davies et al . , 2002 ; pollock and meltzer , 2002 ) .
this gain of function mutation is a valine to glutamic acid substitution at codon 600 , which results in a hyper - activated kinase and subsequent marked increases in mapk / erk signaling ( wan et al . , 2004 ) .
increased b - raf v600e activity is demonstrated to promote bim phosphorylation , leading to its proteasome - mediated degradation in several melanoma models ( cartlidge et al .
, 2008 ; sheridan et al . , 2008 ; boisvert - adamo et al . , 2009 ; goldstein et al . , 2009 ) .
the b - raf v600e mutation is observed very early in melanoma development starting at the benign nevus stage .
therefore , reducing bim levels and all apoptotic pathways that bim directly regulates ( e.g. , cytokine / growth factor withdrawal ) likely leads to marked resistance to apoptosis ( bouillet et al . , 1999 ) . likewise
, since bim is a direct activator of bax and bak , chemotherapies that utilize a bim - dependent mechanism will likely not be successful ( letai et al . , 2002 ; kuwana et al . , 2005 ; dai et al . , 2008 ) .
multiple other pro - apoptotic bcl-2 proteins are also targeted by the b - raf v600e mutation .
several studies suggest that excessive signaling through b - raf and mapk promotes bad ( bcl-2 antagonist of cell death ) phosphorylation and inactivation , and decreased bmf ( bcl-2 modifying factor ) and puma expression , and all these events lead to abrogated bak / bax activation , momp , and apoptosis ( cartlidge et al .
, 2009 ; goldstein et al . , 2009 ; keuling et al . , 2010 ; shao and aplin , 2010 ) .
there is also a growing literature connecting ras and b - raf mutations to the induction of autophagy .
autophagy is a catabolic process which promotes the degradation of cytoplasm and organelles ; and this plays a key role in tumorigenesis as it often helps cells cope with stress ( for in - depth review , see kondo et al . , 2005 ) .
activating mutations in ras and b - raf promote autophagy , which can restrict tumor cell growth ( chen and karantza - wadsworth , 2009 ; maddodi et al . , 2010 ; elgendy et al . , 2011 ) .
in contrast , some tumor cells display an addiction to autophagy , which can promote glucose metabolism and mitochondrial function , leading to enhanced tumorigenesis ( guo et al . , 2011 ; lock et al . , 2011 ) .
interestingly , anti - apoptotic bcl-2 proteins function at the intersection between autophagy and apoptosis by directly regulating beclin-1 activity , a key protein that is required for the initiation of the formation of the autophagy machinery .
therefore , integrating the mechanistic interplay between the pathways that promote melanoma development , autophagy , and the cell death machinery is of critical importance to understanding this disease and potential therapeutic strategies ( levine et al . , 2008 ; chipuk et al . ,
importantly , the regulation of anti - apoptotic bcl-2 proteins by bh3-only proteins and bh3 mimetics ( discussed later ) impacts not only on apoptosis , but also autophagy , and subsequent changes to stress signaling , proteostasis , and metabolism ; yet we will focus our discussion on cellular commitment to apoptosis .
the role of the anti - apoptotic bcl-2 proteins in melanoma is highlighted by the phenotype of the bcl-2 knockout mouse which displays dramatic graying of the hair because of diminished follicular melanocyte survival ( yamamura et al . , 1996 ) .
in support of this phenotype , microphthalmia associated transcription factor ( mitf ) , a key transcription factor necessary for melanocyte development and survival , directly induces bcl-2 promoter activity ( mcgill et al . , 2002 ) .
elevated levels of bcl-2 and mitf can promote chemoresistance and are associated with melanoma progression ; furthermore , antisense against bcl-2 or mitf promotes a decrease in melanoma cell survival and sensitivity to chemotherapy ( jansen et al .
perhaps this cell type does not express other anti - apoptotic bcl-2 proteins , so small increases in bh3-only protein activity may promote premature momp and apoptosis , whereas this normally tolerated level of bh3-only protein activity would be actively sequestered by bcl-2 to allow for stress resolution .
such a limited repertoire of anti - apoptotic reserve would not appear to favor chemoresistance .
however , the malignant transformation and chemoresistance seen in melanomas has also been attributed to increased levels in a1 ( bcl-2 related gene a1 ) , bcl - xl , and mcl-1 , allowing for a rather substantial shift in the apoptotic threshold ( tang et al . , 1998 ; leiter et al . , 2000 ; zhang and rosdahl , 2006 ; placzek et al . , 2010 ; zhuang et al . , 2010 ) .
the upregulation of these anti - apoptotic proteins during the transition from nevus to melanoma may indicate their active role in melanoma progression ( wong et al .
several additional transcription factors such as ets-1 ( v - ets erythroblastosis virus e26 oncogene homolog 1 ) , and the chromatin remodeling factor dek , promote mcl-1 expression , and may present novel mechanisms ( and perhaps chemotherapeutic strategies ) as to the mechanism of melanoma survival and chemoresistance ( khodadoust et al . , 2009 ; dong et al .
. from both in vitro and in vivo melanoma model systems , several lines of evidence suggest that bcl-2 over - expression correlates with a malignant phenotype and a higher metastatic potential ( grover and wilson , 1996 ; takaoka et al . , 1997 ;
leiter et al . , 2000 ; utikal et al . , 2002 ; zhang and rosdahl , 2006 ) .
this seems logical , as a cell expressing more anti - apoptotic proteins should resist apoptosis despite cues to die following events from benign status to malignancy and metastasis .
however , there are conflicting results in the literature that describe decreased bcl-2 expression during melanoma progression ( saenz - santamaria et al . , 1994 ; ramsay et al . , 1995 ; tron et al .
, 1995 ; tang et al . , 1998 ; korabiowska et al . , 1999 ;
similarly , bcl-2 is described to be constitutively expressed in melanocytes , nevi , and melanoma , with little change in expression levels ( cerroni et al . , 1995 ; collins and white , 1995 ; morales - ducret et al . , 1995 ; plettenberg et al . , 1995 ;
the observations in both these scenarios can be reconciled if we consider that a cell may have the opportunity to express alternative anti - apoptotic bcl-2 members , such as bcl - w , bcl - xl , mcl-1 , or a1 .
another possibility is downregulation of the pro - apoptotic effectors bak and bax , which is also associated with tumor progression and decreased patient survival ( fecker et al . , 2006 ; tchernev and orfanos , 2007 ) .
the balance between pro - apoptotic and anti - apoptotic bcl-2 members could also be considered to control apoptosis .
for example , the bax / bcl-2 ratio in chemotherapy - sensitive cells is relatively higher than in cells displaying chemoresistance ( raisova et al . , 2001 ) .
however , great caution should be employed with monitoring only a few proteins , as momp and apoptosis proceed when multiple bh3-only proteins collaborate to promote bak / bax activation .
understanding the dynamic and total proteinprotein interactions within the bcl-2 family is more indicative of apoptotic sensitivity and chemotherapeutic responses .
this has been recently applied to tumors of lymphoid origin , and should be developed to better delineate which patients have the best chance of chemotherapeutic responses ( deng et al . , 2007 ; ryan et al .
returning to our original questions , it appears that melanoma has developed numerous strategies to block apoptosis despite heightened cellular proliferation cues : the loss of bh3-only protein function , along with collateral increases in anti - apoptotic bcl-2 protein expression and diversity .
the marked increase in anti - apoptotic bcl-2 proteins suggests that these cells can engage a pro - apoptotic response , and that the increased bcl-2 expression favors a selection process where increased bcl-2 ( or other anti - apoptotic members ) confers a survival advantage .
strategies that promote bh3-only protein function , while also decreasing the functional anti - apoptotic bcl-2 repertoire , will likely yield the best clinical responses ; indeed , we will discuss the results of such strategies in the following sections .
standard treatments for patients diagnosed with stage iv metastatic malignant melanoma are dtic and il-2 .
the fda approved dtic in 1975 , and it remains the most commonly used single agent chemotherapeutic for metastatic melanoma .
unfortunately , the response rate is less than 20% and resistance nearly always occurs ( stein and brownell , 2008 ) .
other chemotherapeutic agents are currently being studied to improve late stage melanoma treatment but few are proving to be more effective than dtic .
for example , temozolomide , a imidazotetrazine - derivative of dtic , was tested in clinical trials but did not improve the overall survival of patients compared to dtic ( patel et al . , 2011 ) .
in addition to the standard combination chemotherapy regimens [ e.g. , cvd ( cisplatin , vinblastine , dtic ) , dartmouth ( dtic , cisplatin , carmustine , and tamoxifen ) , and bold ( bleomycin , vincristine , lomustine , and dtic ) ] , a number of novel adjuvant therapies are currently being evaluated to improve the efficiency of dtic - based treatments ( seigler et al . , 1980 ; legha et al . , 1989 ; lattanzi et al . , 1995 ) .
in particular , ipilimumab , a human igg1 monoclonal antibody that recognizes cytotoxic t - lymphocyte - associated antigen 4 ( ctla-4 ) , enhances t - cell activation , proliferation , and attack of cancer cells ( melero et al .
fong and small , 2008 ; robert and ghiringhelli , 2009 ; hodi et al . ,
2010 ) . the combination of dtic and ipilimumab was recently shown to be associated with a significant increase in survival among patients with untreated metastatic melanoma ( robert et al . , 2011 ) .
the mechanism of action for dtic - based therapies is to kill tumor cells by dna damage , and these treatments likely signal through the p53 pathway to induce apoptosis ( igney and krammer , 2002 ; vousden and lane , 2007 ) .
fortunately , p53 is rarely targeted in melanoma , so the pro - apoptotic arm of the p53 pathway has the potential to be engaged .
dtic also directly impacts on the bcl-2 family from several angles : there is downregulation of anti - apoptotic proteins , and coordinated upregulation of bh3-only proteins and effector molecules , like bim and bax , respectively ( weber et al . , 2009 ; jiang et al . , 2010b )
importantly , dtic has been studied in multiple cellular models of melanoma , and the consistent observation is that dtic promotes apoptosis through a marked regulation of the bcl-2 family leading to momp . but what accounts for the low response rate of melanoma patients to dtic ? can the threshold for bak / bax activation , momp , and apoptosis be lowered to increase dtic responses in patients ? there is minimal information regarding how combination chemotherapy regimens ( e.g. , cvd ) impact on the bcl-2 family .
it is therefore essential to better understand the mechanisms of action to design appropriate co - administered adjuvant therapies .
a non - exhaustive list of several chemotherapeutic drugs ( some discussed below ) and their impact on the bcl-2 family in common melanoma cell lines is provided in table 1 . a list of chemotherapeutics that induce changes to the bcl-2 family in various melanoma cell lines . in terms of a targeted therapeutic approach
, the frequency of the b - raf v600e mutation in melanoma presents a novel and unique drug target ( davies et al . , 2002 ; sheridan et al . , 2008 ) .
early studies with b - raf v600e mutated melanoma lines suggest that silencing b - raf v600e expression leads to greater sensitivity to apoptosis , which is likely mediated by the combined stabilization and function of bim to promote bak / bax activation
in recent years , treatment of melanoma patients harboring the b - raf v600e mutation with small molecules ( e.g. , plx-4720 , plx-4032 ) that specifically inhibit the b - raf v600e kinase has been successful through phase iii clinical trials , and appears to offer greater response rates and increased overall survival compared to dtic ( flaherty et al . , 2010 )
. however , melanoma cells treated with these drugs have a means of becoming resistant to declining levels of b - raf signaling . for instance , if pten expression or function is lost , there are suggestions in the literature that melanoma cell lines can develop resistance to plx-4720 induced death due to decreased bim ( paraiso et al . , 2011 ) .
the loss of pten results in constitutive activation of the pi3k / akt pathway , which has been described in melanoma suggesting b - raf inhibitors will only work for a subset of patients carrying the b - raf v600e mutation and functional pten ( vanbrocklin et al . , 2009 ) .
dual requirements for the pi3k / akt pathway and constitutive mapk signaling are also supported by combination treatments such as sorafenib [ small molecular inhibitor of several tyrosine protein kinases ( vegfr and pdgfr ) and b - raf ] and nanoliposomal ceramide , which have been shown to synergistically decrease pi3k / akt and mapk signaling leading to increased sensitivity to the mitochondrial pathway of apoptosis in vitro ( tran et al . ,
furthermore , several mek pathway inhibitors , such as uo126 and ci-1040 , have also been shown to cause upregulation of pro - apoptotic bcl-2 protein function like bim , bmf , noxa , and puma , and occasionally can lead to the downregulation of anti - apoptotic proteins like bcl-2 and mcl-1 ( wang et al . , 2007 ; vanbrocklin et al . , 2009 ) .
perhaps combinations of b - raf and mek inhibitors can promote more robust apoptosis due to dual regulations of numerous anti- and pro - apoptotic bcl-2 members ( wang et al . , 2007 ) .
from our discussion , we suggest that melanoma likely develops because of altered bcl-2 family function , which allows for survival and proliferation despite signals to die . likewise , literature supports that chemotherapies engage apoptosis by directly promoting the function of pro - apoptotic bcl-2 members .
if so , are there other drugs that can be used in combination with dtic to alter the balance toward death , perhaps by decreasing anti - apoptotic bcl-2 protein expression , or by inhibiting their function by inducing bh3-only proteins ? indeed , there is already an abundant literature to show that many drugs can directly regulate the bcl-2 family to favor turning on the mitochondrial pathway of apoptosis ( table 1 ) .
numerous dna damaging agents , such as cisplatin , directly impinge on the mitochondrial pathway of apoptosis by lowering anti - apoptotic bcl-2 protein expression , while also increasing effector levels , and presumably , activation and momp ( shibuya et al .
endoplasmic reticulum stress induced by tunicamycin or thapsigargin can also induce apoptosis of melanoma cells , which has been shown to directly up - regulate puma expression ( jiang et al . , 2008 ) .
proteasome inhibitors such as bortezomib have been shown to induce bid , noxa , and puma in melanoma cells ( nikiforov et al .
drugs with diverse mechanisms of action including dihydroartemisinin ( promotes oxidative stress ) , adi - peg20 ( arginine deiminase ) , and aminooxyacetate ( transaminase inhibitor ) appear to directly regulate noxa expression , which has been shown to directly impact on cellular sensitivity to both intrinsic and extrinsic forms of apoptosis ( qin et al .
the overall outcome of such studies suggests that the bcl-2 family determines the chemotherapeutic response to either engage apoptosis or continue with survival .
indeed , several drugs are suggested to promote apoptosis via bim , which nicely parallels existing clinical data that shows melanoma progression and poor survival rates are associated with decreased bim expression ( cartlidge et al .
are there strategies to sensitize melanoma cells to chemotherapeutics by using this information ? in the following section , we provide evidence that depleting cellular anti - apoptotic bcl-2 function may be the optimal combination therapy to promote chemotherapeutic responses despite over - expressed anti - apoptotic bcl-2 members or decreased pro - apoptotic bcl-2 engagement .
as mentioned , the bcl-2 family of proteins can influence both the progression and chemotherapeutic responses of melanoma . while conventional chemotherapeutic regimens can modulate the levels of individual anti- and/or pro - apoptotic bcl-2 proteins , changes in one or two proteins
furthermore , since anti - apoptotic proteins are numerous , there are multiple members with the potential to inhibit pro - apoptotic signaling leading to chemoresistance . while there are notions to suggest that loss of bid / bim or bak / bax can lead to cancer development and/or chemoresistance , most data suggest that these proteins are not commonly targeted , which affords the over - expressed anti - apoptotic members a unique opportunity for pharmacological regulation ( letai , 2008 ) . with this understanding , directly targeting the anti - apoptotic bcl-2 proteins , and indirectly the pro - apoptotic machinery they control ,
is an attractive tool with potential to generate new therapies , as well as complement existing treatments . in this endeavor ,
the greatest successes have come through the identification of small molecules that mimic the function of bh3-only proteins to bind within the hydrophobic pocket of the anti - apoptotic bcl-2 proteins .
these small molecules , referred to as  bh3 mimetics ,  functionally neutralize several anti - apoptotic proteins , thus inhibiting their ability to sequester pro - apoptotic proteins .
this strategy lowers the cellular threshold leading to bak / bax activation , momp , and subsequent apoptosis .
the notion that regulating bcl-2 function in melanoma could yield enhanced responses to chemotherapeutics arose from a study that showed bcl-2 antisense improved treatment responses and tumor burden in a xenograft mouse model of human disease ( jansen et al . ,
however , the use of bcl-2 antisense adjuvant therapy ( oblimersen sodium ) with dacarbazine failed to meet expectations in several trials ( conducted by genta incorporated ) , although there continues to be some controversy as to whether or not bcl-2 antisense is beneficial ( jansen et al .
independent of those results , pharmacological regulation of anti - apoptotic bcl-2 proteins continues to be of tremendous interest .
a bh3 mimetic that has been studied with considerable success in melanoma is the abbott compound abt-737 ( and the bio - available form abt-263 )
. published in 2005 , abt-737 was identified using a structural activity relationship by nuclear magnetic resonance ( sar by nrm ) screen for compounds that could bind within the hydrophobic groove of bcl - xl ( oltersdorf et al . , 2005 ) .
upon its discovery , abt-737 was determined to have high affinity for the anti - apoptotic bcl-2 proteins bcl-2 , bcl - w , and bcl - xl , but not a1 or mcl-1 .
abt-737 was immediately cast as a promising new drug candidate since it could engage multiple anti - apoptotic bcl-2 family members , and it demonstrated minimal toxicity as a single agent .
however , the inability of abt-737 to inhibit mcl-1 ( or a1 , however , a1 has restricted expression so the influence in cancer is far less than mcl-1 ) , which is commonly over - expressed in melanomas , highlighted one weakness for using abt-737 in melanoma , and suggested a possible mechanism by which abt-737 resistance may be conferred ( yecies et al . , 2010 ) .
indeed , melanoma survival and sensitivity to chemotherapeutics appears to be mediated by a combination of anti - apoptotic bcl-2 members as preclinical studies using antisense oligonucleotides against bcl-2 , bcl - xl , or mcl-1 sensitized to chemotherapy - induced apoptosis ( gautschi et al .
, 2001 ; del bufalo et al . , 2003 ; thallinger et al . ,
a recent study examined the result of reduced mcl-1 expression in combination with abt-737 treatment in melanoma cells ( keuling et al . , 2009 ) .
indeed , the results showed an increased induction of apoptosis , and opened up the possibility for pro - apoptotic synergy between abt-737 and classical chemotherapeutic treatments that decrease mcl-1 levels .
for example , abt-737 in combination with the standard clinically approved anti - melanoma drug , dacarbazine , promoted apoptosis through the induction of noxa , which likely synergizes with abt-737 to fully inhibit the anti - apoptotic repertoire ( weber et al . , 2009 ) . in a similar scenario , abt-737 synergized with the proteasome inhibitor mg-132 , which was also suggested to induce noxa expression and inhibition of mcl-1 function leading to increased rates of apoptosis ( fernandez et al . , 2005 ; qin et al . , 2005 ; miller et al . , 2009 )
. alternatively , because of frequent mutations in the mapk pathway in melanoma , abt-737 has also been studied in combination with mek and p38 inhibitors , both of which synergize to enhance the apoptotic machinery ( cragg et al . , 2008 ; keuling et al .
tw-37 is another bh3 mimetic , but it has a broader range of targets including bcl-2 , bcl - xl , and mcl-1(verhaegen et al . , 2006 ) . due to the ability of tw-37 to inhibit multiple members , in particular mcl-1 ,
it was proposed that tw-37 could synergize with chemotherapeutics to promote apoptosis independently of noxa regulation .
indeed , when melanoma lines were treated with tw-37 and a mek inhibitor ( u0126 ) , significant apoptosis resulted .
interestingly , the observed synergy did not occur in normal melanocytes , suggesting that the mechanism of action was specific to tumor cells .
curiously , the observed apoptosis was also dependent upon the p53 pathway , which is functionally intact in the majority of melanoma cell lines and patients .
obatoclax is also an inhibitor to the anti - apoptotic bcl-2 family proteins that has been studied in melanoma .
first described in 2007 , obatoclax is believed to inhibit all of the anti - apoptotic bcl-2 proteins , including mcl-1 , and thus shows less resistance to treatment than abt-737 ( nguyen et al .
while obatoclax has been shown to induce apoptosis as a single agent treatment in other types of cancer , such efficacy has not been observed in melanoma , leading to the study of combination treatments ( trudel et al . , 2007 ) .
when combined with the endoplasmic reticulum stressors tunicamycin or thapsigargin , increased apoptosis was observed ( jiang et al . , 2009 ) .
this synergy is likely due to obatoclax functioning like a sensitizer bh3-only protein to inhibit the anti - apoptotic bcl-2 proteins , lowering the cellular threshold for bak / bax activation , and allowing the mitochondrial apoptotic machinery to be engaged when signaled .
however , it should be noted that despite its confirmed inhibiting interaction with the anti - apoptotic bcl-2 proteins , albeit at a higher concentrations than abt-737 , the mechanism by which obatoclax elicits a response is not definitive , as studies have suggested alternative targets than the bcl-2 family ( konopleva et al . , 2008 ) . while the above drugs target the anti - apoptotic bcl-2 proteins , none of them have proven to be as effective at binding the anti - apoptotics proteins as the bh3-only proteins themselves .
given the importance of the bh3 domain  anti - apoptotic interaction , which lowers the cellular threshold of apoptosis , short chemically synthesized peptides mimicking the different bh3-only proteins have been generated and tested for function ( letai et al . , 2002 ) .
indeed , bh3 domain peptides from different bh3-only proteins revealed the distinction between direct activator and sensitizer / de - repressor bh3-only proteins ( figure 3 ) . the bid and bim bh3 domain peptides promote bak / bax - dependent momp and cytochrome c release , whereas the bad bh3 promotes momp by inhibiting bcl-2 , bcl - xl , and bcl - w ( and revealing bid / bim activity , see figure 3 ) , and not by direct bak / bax interactions ( chen et al .
one issue that arose with using synthetic peptides was the loss of -helicity that is required for protein - protein interactions between the bh3-only proteins and the anti - apoptotics . to address this issue , non - natural amino acids containing olefin - bearing tethers
were introduced into the bh3 domain sequence , effectively  stapling  the peptide into an -helical form ( walensky et al . , 2004 , 2006 ) .
these stapled bh3 domains are referred to as  stabilized -helix of bcl-2 domains  ( sahbs ) , and these may serve as a new tool for designing cancer therapeutics to target the bcl-2 family and enhance endogenous bh3-only activity .
for example , the bid bh3 sahb was shown to induce apoptosis in leukemia cells as well as inhibit growth of human leukemia xenografts in vivo ( walensky et al . , 2004 ) .
recently , a mcl-1 bh3 sahb was developed which inhibits mcl-1 with very high affinity , and indeed has also been shown to sensitize cancer cells to apoptosis ( n.b . , the mcl-1 sahb uniquely binds to the mcl-1 hydrophobic groove ; stewart et al . ,
the identification of an exclusive and potent mcl-1 inhibitor is important , given the lack of potent inhibitors that target mcl-1 and the emergence of mcl-1 as a chemoresistance factor in a broad range of human cancers .
these developments will hopefully serve as an additional platform to discover small molecule regulators of the bcl-2 family .
presently , bh3 domain peptides have not been examined in the context of melanoma treatment ; efforts to elucidate their potential impact would be useful for understanding both the biology of melanoma , and hopefully its treatment . of note ,
bh3 mimetics also promote autophagy , alone or in combination with b - raf inhibition ; and this may be a contributing mechanism with therapeutic potential in metastatic melanoma ( armstrong et al . , 2011 ; malik et al .
throughout our discussions , we focused on applying the mechanisms of action for the bcl-2 family to the regulation of melanoma tumorigenesis , apoptosis , and treatment . as the bcl-2 family defines the balance between life and death in normal and cancer cells , it is reasonable to seek a full understanding of the cellular mechanisms that control bcl-2 family function to establish how stressed cells undergo apoptosis , and how cancer cells resist pro - apoptotic signals to ensure survival .
here we suggest that multiple anti - apoptotic bcl-2 proteins ensure survival , and that melanocytes undergoing stress during tumorigenesis and metastasis increase the expression and/or function of these pro - survival members .
likewise , chemotherapeutics ( single agents and combinations ) must promote the inhibition of these pro - survival members by a combination of bh3-only protein functions to engage bak / bax activation and achieve desired clinical outcomes . while we have focused on the direct targeting of the bcl-2 family to promote momp , it is important to mention that there are also pharmacological opportunities to promote apoptosis both upstream and downstream of mitochondria . in particular , the development of xiap ( x - linked inhibitor of apoptosis protein ) inhibitors is critical as the increased expression of xiap is positively correlated with melanoma thickness , tumor progression , and chemoresistance ( chawla - sarkar et al . , 2004 ; kluger et al .
xiap is a potent inhibitor to the apoptotic caspases , so relieving this apoptotic brake is predicted to lower the threshold leading to the cell death phenotype ( eckelman et al . , 2006 ) .
indeed , the development of smac ( second mitochondria - derived activator of caspases , the cellular xiap inhibitor ) mimetics to target xiap is shown to induce apoptosis in numerous melanoma cell lines ( vucic et al . , 2002
there are also numerous additional pathways that offer opportunities to enhance tumor cell killing though momp and apoptosis .
for example , epigenetic mechanisms are implicated in melanoma survival as mir-149 and mir-193b enhance mcl-1 expression in nevi , patient tumor samples , and melanoma cell lines ( chen et al . , 2011 ; jin et al . , 2011 ) .
histone deacetylase ( hdac ) inhibitors sensitize melanoma cells to chemotherapy through a number of mechanisms , including suppression of the ras / mapk signaling pathway and enhanced dna damage signaling leading to apoptosis ( peltonen et al . , 2005 ; kobayashi et al . , 2006 ; munshi et al . ,
furthermore , the role of chromatin modifications may offer collateral means of pharmacological intervention as the requirement for swi / snf complexes in mitf regulated melanocyte - specific gene expression pathways directly impact on bcl-2 expression , and likely sensitivity to chemotherapeutics ( de la serna et al .
2010 ) . determining the appropriate combination treatments for each patient based on his / her individual tumor status will likely generate the most effective treatments
fortunately , the cellular signaling pathways that intersect between the survival and apoptosis cascades are numerous ( e.g. , b - raf mutation and bcl-2 over - expression ; proteasome function and mcl-1 over - expression ) and this affords the pharmacological opportunity to explore multiple combination strategies to cure melanoma . in the future , enhanced animal models of human disease will likely shed light upon the mechanisms that drive melanocyte tumorigenesis and metastasis .
the combined b - raf v600e / pten mouse model mentioned earlier is perhaps the ideal model system to genetically evaluate the role of the bcl-2 family in melanoma progression ( dankort et al . , 2009 ) .
as further models are developed , they should be evaluated along side the standard models of bcl-2 family function ( e.g. , bak 
 bax , bidbim , puma , melanocyte - specific bcl-2 and mcl-1 transgenics , etc  ) to fully appreciate the influence of the bcl-2 family in mediating melanoma development and treatment . likewise ,
a better understanding of the pathways ( e.g. , mitochondrial dynamics , structural studies , lipid environment etc  ) that regulate the bcl-2 family itself will certainly yield novel target strategies to obtain better clinical outcomes .
the future shows significant promise to develop more specific and bio - available small molecule inhibitors to the anti - apoptotic bcl-2 proteins , and perhaps drugs that directly promote bak / bax activation ; this will be another significant milestone in adjuvant - based chemotherapeutic strategies targeting the mitochondrial pathway of apoptosis .
the hope is that as we further dissect the mechanisms of the bcl-2 family and apoptotic pathways , novel therapeutic targets and combination strategies will emerge and provide significant benefits to those afflicted with melanoma .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 the global incidence of melanoma has dramatically increased during the recent decades , yet the advancement of primary and adjuvant therapies has not kept a similar pace . the development of melanoma is often centered on cellular signaling that hyper - activates survival pathways , while inducing a concomitant blockade to cell death . 
 aberrations in cell death signaling not only promote tumor survival and enhanced metastatic potential , but also create resistance to anti - tumor strategies . 
 chemotherapeutic agents target melanoma tumor cells by inducing a form of cell death called apoptosis , which is governed by the bcl-2 family of proteins . 
 the bcl-2 family is comprised of anti - apoptotic proteins ( e.g. , bcl-2 , bcl - xl , and mcl-1 ) and pro - apoptotic proteins ( e.g. , bak , bax , and bim ) , and their coordinated regulation and function are essential for optimal responses to chemotherapeutics . here 
 we will discuss what is currently known about the mechanisms of bcl-2 family function with a focus on the signaling pathways that maintain melanoma tumor cell survival . 
 importantly , we will critically evaluate the literature regarding how chemotherapeutic strategies directly impact on bcl-2 family function and offer several suggestions for future regimens to target melanoma and enhance patient survival . 
Introduction
The mitochondrial pathway of apoptosis
The BCL-2 family of proteins
Melanoma and the BCL-2 Family
Melanoma Treatment and the BCL-2 Family
BCL-2 Family Inhibitors and Melanoma Treatment Responses
Perspectives
Conflict of Interest Statement