Status of Aurora kinase A and Aurora kinase B expression in nevus and melanoma tissues and melanoma cell lines
Probe sets from a whole-genome microarray analysis, which we previously performed,2
of cryopreserved normal skin, benign nevi, atypical nevi, which are the precursors and risk markers of melanoma, and melanomas in situ
, which although noninvasive, are the first stage of melanoma development, VGP and MGP melanomas, and melanoma-infiltrated lymph nodes, provided a first indication that the Aurora kinases A and B are upregulated with progression from early to advanced melanoma (). This observation prompted us to probe 1) cryopreserved tissue specimens, ranging from normal skin all the way to melanoma-infiltrated lymph nodes; 2) a nevus > melanoma progression tissue microarray (TMA), comprised of more than 180 tissue cores; and 3) tissue sections from randomly selected formalin-fixed, paraffin-embedded (FFPE) melanoma specimens with an antibody to Aurora kinase A and, likewise, an antibody to Aurora kinase B. With the exception of some epidermal keratinocytes and/or dermal fibroblasts in normal skin, benign and atypical nevi, and melanoma in situ
that stained positive for Aurora kinase B, the cryopreserved tissues exhibited little expression of Aurora kinase B () or Aurora kinase A (data not shown). In contrast, Aurora kinase B and likewise Aurora kinase A (data not shown) were strongly expressed in cryopreserved tissue samples representing VGP and MGP melanomas and melanoma-infiltrated lymph nodes (LN) (). Scored on a signal-intensity scale of 0 > 3, the nevus > melanoma progression TMA analysis yielded very similar results (data not shown). In addition, the TMA data revealed that the number of VGP, MGP, and LN melanoma tissue cores that demonstrated expression of Aurora kinase B was 5-fold higher than the number of Aurora kinase A–positive melanoma tissue cores (data not shown). Depicted in are examples of an MGP melanoma TMA core and 2 adjacent tissue sections of a randomly selected FFPE MGP melanoma specimen, probed with Aurora kinase A, and likewise Aurora kinase B antibody.
Figure 1. Expression of Aurora kinases A and B in normal skin and nevus and melanoma tissue specimens subjected to whole-genome microarray analysis. Levels of Aurora kinase A (orange-colored bars) and Aurora kinase B (green-colored bars) expression in cryopreserved (more ...)
Figure 2. Aurora kinase A and Aurora kinase B expression in cryopreserved and archival nevus and melanoma tissues and VGP and MGP melanoma cell lines. (A) Cryopreserved tissue sections, prepared from normal human skin (NS), a benign (BN) and an atypical nevus (AN), (more ...)
In addition to these tissues, we also analyzed VGP and MGP melanoma cell lines for the status of Aurora kinase A and Aurora kinase B expression. RT-PCR analysis of 2 MGP melanoma cell lines (WM1158 and WM983-B) with a human Aurora kinase B–specific set of primers led to the amplification of a single 302-bp Aurora kinase B transcript (, panel a), and immunoblot analysis of 2 VGP (WM983-A and WM98-2) and 4 MGP melanoma cell lines (WM373, WM852, WM983-B, and WM1158) demonstrated the presence of Aurora kinase A (, panel b) and Aurora kinase B protein (, panel c) in every one of these cell lines.
Downregulating the expression of Aurora kinase A or B leads to inhibition of melanoma cell proliferation
Using a pool, comprised of 4 Aurora kinase A and likewise 4 Aurora kinase B–specific siRNAs, we transfected WM1158 MGP melanoma cells, which as determined by immunoblot analysis led to downregulation of Aurora kinase A (, panel a) and, similarly, Aurora kinase B expression (, panel b) at 24, 48, and 72 hours following transfection. In addition, phosphorylation of the Aurora kinase B substrate, Ser10 on histone 3 (pHisH3), was reduced starting at 48 hours following transfection with the Aurora kinase B–specific siRNAs (, panel c). Furthermore, starting at 48 hours, and becoming more apparent thereafter, the proliferation of the Aurora kinase A and similarly, albeit less pronounced, the Aurora kinase B siRNA-transfected WM1158 MGP melanoma cells was inhibited compared with the proliferation of WM1158 cells that, serving as controls, had received only the siRNA delivery vehicle, Lipofectamine, or were transfected with a pool of 4 nontargeting siRNAs ( and ).
Figure 3. Immunoblot and cell proliferation analysis of Aurora kinase A and Aurora kinase B siRNA-transfected MGP melanoma cells. (A) Total cell lysates of WM1158 MGP melanoma cells, transfected with (a) 150 nM Aurora kinase A siRNAs (siRNA) or (b and c) 150 nM (more ...)
Treatment of melanoma cells with an Aurora kinase small-molecule inhibitor leads to overt changes in melanoma cell morphology and cell division
To determine whether in addition to inhibiting expression of the Aurora kinases A and B, blocking the function of these 2 molecules would interfere with the biological features of advanced melanoma, we obtained the Aurora kinase small-molecule inhibitor, PF-03814735, whose IC50
value (nmol/L) for Aurora kinase A is 5 ± 3 and for Aurora kinase B is 0.8 ± 0.6.9
Using as a first step the concentrations of 1 nM and 10 nM as well as 0.1 µM, 1 µM, and 10 µM, we found that starting at 1 µM and becoming most pronounced at 10 µM, VGP (WM983-A) and several MGP melanoma cells, including the WM1158 MGP melanoma cells (, panel c), rapidly severed their cell-cell contacts, in some cases formed long dendrites, a process indicative of onset of terminal differentiation, and starting at about 72 hours following addition of the Aurora kinase small-molecule inhibitor, massively dislodged into the growth medium. Furthermore, cells that had detached from the surface of the Petri dish and dislodged into the growth medium did not reattach to a tissue culture dish after they had been rinsed several times with complete growth medium not containing the inhibitor.
Figure 4. Aurora kinase inhibitor treatment of MGP melanoma cells. (A) Morphology of MGP melanoma cells (WM1158) not treated (panel a), that received only DMSO (panel b), or were treated with 10 µM of Aurora kinase inhibitor (panel c) for 24 or 48 hours. (more ...)
To determine to which extent this small-molecule inhibitor when added to melanoma cells blocked primarily the function of the 2 Aurora kinases, we pursued a series of immunoblot and optical imaging studies. Like in the case of almost all small-molecule inhibitors, PF-03814735 has been reported to inhibit, in addition to Aurora kinase A and B, other molecules including Flt1, FAK, TrkA, Met, and FGFR-1, albeit with significantly lower affinity.9
However, we did not obtain experimental evidence that, for example, FGFR-1, which correlating with melanocytic progression is upregulated to high levels in advanced melanoma,10,11
was not or no longer phosphorylated in melanoma cells treated with the PF-03814735 inhibitor (). In contrast, treatment of melanoma cells for 1 hour with 1 µM or 10 µM of the inhibitor revealed that the kinase activity of Aurora kinase A and phosphorylation of Ser10 on histone 3 were impaired (, lanes c and d). Similarly, Aurora kinase A was no longer phosphorylated when the cells were treated with 10 µM of the inhibitor for 24 hours (, lane c) or 48 hours (, lane f). In addition, immunoblot analysis of WM1158 MGP melanoma cells incubated in the presence of nocodazole for 20 hours, followed by addition of 10 µM of the Aurora kinase small-molecule compound for 5, 10, or 60 minutes, demonstrated that Ser10 on histone H3 was no longer phosphorylated at 60 minutes posttreatment (, lane i).
Immunofluorescence imaging of WM1158 MGP melanoma cells that had been treated with the Aurora kinase inhibitor for 2 hours and then were probed with an antibody to Aurora kinase A pT288 as well as an antibody to α-tubulin (, panel b), or that had been incubated in the presence of nocodazole and thereafter were treated for 2 hours with the inhibitor and then stained with an antibody to pHisH3 as well as an α-tubulin antibody (, panel b), revealed substantial perturbation of the microfilament structure when compared to cells that were not treated with the inhibitor ( and , panel a). Furthermore, immunofluorescence imaging of nocodazole-treated WM1158 MGP melanoma cells that were treated for 2 hours with the Aurora kinase inhibitor and then probed with antibody to CREST to mark kinetochores, Aurora kinase A, Aurora kinase B, as well as α- or y-tubulin demonstrated disruption of the spindle checkpoint (, panels b, d, f, h) compared to WM1158 MGP melanoma cells that had not been treated with the small-molecule agent (, panels a, c, e, g).
Blocking the function of Aurora kinase A and B inhibits melanoma cell proliferation and causes melanoma cell cycle dysregulation and apoptosis
To determine whether, as in the case of downregulating the expression of the Aurora kinases by way of RNA interference, interfering with their functions would lead to inhibition of melanoma cell proliferation, we treated MGP melanoma cells with the Aurora kinase inhibitor for up to 5 days. As shown in , starting as early as 24 hours posttreatment, the proliferation of the melanoma cells was markedly inhibited and to a significantly greater extent than in the prior experimental setting where we had suppressed via siRNAs and the expression of Aurora kinase A and likewise of Aurora kinase B ( and ).
Figure 5. Inhibiting the function of Aurora kinases A and B leads to inhibition of melanoma cell proliferation, dysregulation of the melanoma cell cycle, and melanoma cell apoptosis. (A) Proliferation of WM1158 MGP melanoma cells at various time points following (more ...)
To analyze whether, alongside with blocking the proliferation of melanoma cells, treatment with the Aurora kinase inhibitor also interfered with the cells’ progression through the cell cycle, we pursued experiments that involved propidium iodide as well as annexin V/propidium iodide–based flow cytometry. WM1158 MGP melanoma cells that were treated for 72 hours with 10 µM of the Aurora kinase inhibitor and then fixed and labeled with propidium iodide revealed a major accumulation of the cells in sub-G0/G1 (), and flow cytometric analysis of annexin V/propidium iodide–labeled melanoma cells that had been treated for 24 or 48 hours with the small-molecule inhibitor documented that substantially more cells were arrested in the early rather than in the late stage of apoptosis (). Additional experimental evidence, which documented that Aurora kinase inhibitor–treated melanoma cells underwent massive apoptosis, came from an immunoblot analysis, which demonstrated cleavage of PARP to cPARP within 24 hours following addition of the inhibitor to the cells (), and from fluorescent imaging analysis of TUNEL-stained cells ().
In vivo and ex vivo analysis of human melanoma xenografts of nude mice treated with Aurora kinase inhibitor
In light of the extreme resistance of advanced melanoma to standard regimens of treatment, and the fact that, to date, only limited information is available regarding genes that might constitute useful targets for molecular therapy of advanced melanoma, we next undertook a series of preclinical studies to determine whether molecular targeting of Aurora kinase A and/or Aurora kinase B would be efficacious for human MGP melanoma cells grown as subcutaneous tumors in nude mice.
The first set of these in vivo
studies involved systemic treatment of nude mice, bearing WM983-B MGP human melanoma xenografts, with the Aurora kinase inhibitor PF-03814735 administered twice a week intraperitoneally (i.p.) at a dose of 30 mg/kg for a total period for 24 days (). Until about the fifth i.p. injection of the inhibitor on day 14, the tumors did not substantially increase in volume. However, following day 14, it became apparent that the MGP melanoma xenografts in mice that continued to receive systemic treatment with the Aurora kinase inhibitor for another 10 days did grow at a slower rate compared to WM983-B MGP melanoma xenograft-bearing nude mice that were not given injections or that received only the Aurora kinase inhibitor delivery vehicle, dimethyl sulfoxide (DMSO) (). Unlike some other currently available Aurora kinase small-molecule agents, PF-03814735 can be given orally. Thus, we also pursued WM983-B human melanoma xenograft studies that for a period of 24 days involved twice-weekly delivery of the Aurora kinase inhibitor (30 mg/kg) by oral gavage. As a third route of delivery, WM983-B human melanoma xenografts received twice-weekly intratumoral injections of the inhibitor at a dose of 2.5 mg/kg or at a 4-fold higher dose of 12 mg/kg. Both of these latter routes of treatment led to similar tumor-growth impairment (data not shown) as we observed in the case of the systemic i.p. route of delivery. Since extensive in vitro
and in vivo
pharmacokinetic (PK) and pharmacodynamic (PD) studies involving PF-03814735 were previously performed and recently published,9
we did not make PK and PD analyses a particular focus in the setting of this melanoma study. Furthermore, since it had been determined that when the small-molecule inhibitor was administered at a dose of 60 mg/kg, animals exhibited weight loss of >20%,9
we did not explore the impact of treating human melanoma xenograft-bearing mice with doses of PF-03814735 higher than the ones we administered, which were well tolerated by the animals.
Figure 6. Aurora kinase small-molecule inhibitor treatment of human melanoma xenograft-bearing nude mice. (A) Nude mice, bearing subcutaneous WM983-B MGP melanoma xenografts, received the first i.p. injection of Aurora kinase inhibitor (30 mg/kg) or Aurora kinase (more ...)
Since it is unlikely that a small-molecule inhibitor, regardless of its molecular target, when administered as a single agent, will ever be effective to the extent that it will be a cure for patients with advanced melanoma, we next determined whether a combination treatment would further enhance the impact of the Aurora kinase inhibitor on MGP melanoma xenografts. Thus, we administered, in the same setting of these in vivo studies, the Aurora kinase inhibitor (30 mg/kg) in combination with the cytotoxic drug paclitaxel (10 mg/kg), which via binding to tubulin, blocks the disassembly of microtubules. Using a similar schedule of twice-a-week systemic treatment, the inhibitor was injected i.p. followed 24 hours later by i.p. injection of paclitaxel. In comparison with the growth rate of the tumors in the nude mice that had only been treated with the inhibitor, the tumors in the animals that had received the combination treatment of Aurora kinase inhibitor and paclitaxel over a period of 24 days grew noticeably slower, suggesting that the combination treatment was more effective.
Our alternative experimental approach to determine to which extent targeting of Aurora kinase A and B would exhibit efficacy for human melanoma xenografts involved the use of an Aurora kinase A and likewise an Aurora kinase B antisense vector, and in addition, a pcDNA-HA dead kinase Aurora B plasmid.12
One hundred micrograms of each of these 2 Aurora kinase AS plasmids and, likewise, the pcDNA-HA dead-kinase Aurora B construct, which has the lysine at position 106 of Aurora kinase B substituted by an alanine,12
were mixed with the delivery vehicle DC-Chol liposomes and injected twice weekly into WM983-B MGP melanoma xenografts for a period of 2 weeks. The 3 respective controls (data not shown) were tumors that did not receive injections, were injected with a pcDNA plasmid not containing a cDNA, or were given intratumoral injections of a pcDNA-HA Aurora kinase B wild-type plasmid construct.12
Although these 3 different Aurora kinase–targeting vectors were not nearly as effective in slowing the growth of the MGP melanoma xenografts as the Aurora kinase small-molecule inhibitor administered in combination with paclitaxel (), we did find that more prominently than the Aurora kinase A or the Aurora kinase B antisense vector, which block gene expression, the Aurora B dead-kinase (DN) vector, which inhibits the function of Aurora kinase B, did impact the growth of the tumors until about the third intratumoral injection but not thereafter ().
Given the results of these in vivo molecular targeting studies, we next determined the extent to which the systemic i.p. treatment with the small-molecule inhibitor when administered alone or in combination with paclitaxel had blocked Aurora kinase function in the tumor cells. Probed with an antibody to pHisH3, tissue sections prepared from the periphery, as well as the center of human melanoma xenografts that had been resected from tumor-bearing nude mice that had been euthanized within 3 hours following the last i.p. injection of the inhibitor on day 24 (), demonstrated numerous pHisH3-positive melanoma cells in the xenografts from the nude mice that had been injected with the small-molecule inhibitor delivery vehicle, DMSO (, panel a). In contrast, melanoma xenografts from the mice that had been treated systemically with the Aurora kinase inhibitor (, panel b) or with a combination of the inhibitor and paclitaxel (, panel c) did not reveal any pHisH3-positive cells. Furthermore, an immunohistochemical analysis with an antibody to the cell proliferation marker, Ki67, revealed noticeable differences between WM983-B MGP melanoma xenografts from mice that were treated with a combination of the inhibitor and paclitaxel (, panel d) and WM983-B MGP melanoma xenografts from mice that did not receive treatment (, panel e).