Targeting mTORC 1 / 2 with OSI-027 inhibits proliferation and migration of keloid keratinocytes

Keloid is a dermal proliferative disorder characterized by the excessive keratinocyte proliferation and migration. mTOR over-activation is involved in the process. Here, we show that both mTOR complex 1 (mTORC1) and mTORC2 were hyper-activated in keloid-derived primary keratinocytes. OSI-027, an mTOR kinase inhibitor, potently inhibited proliferation and migration of keloid keratinocytes. OSI027 disrupted the assembly of mTORC1 (mTOR-Raptor) and mTORC2 (mTOR-RictormLST8). Further, OSI-027 completely blocked the phosphorylations of the mTORC1 substrates (S6K1, S6 and 4EBP1) and the mTORC2 substrate (AKT, at Ser-473). OSI-027 was potent than rapamycin in inhibiting keloid keratinocytes. Moreover, restoring mTORC1 activation by the introduction of the constitutively active S6K1 only partly alleviated OSI-027-induced suppression on keloid keratinocytes. Notably, mTOR2 inhibition by Rictor siRNA also inhibited keloid keratinocyte proliferation and migration, although less efficiently than OSI-027. Together, concurrent targeting of mTORC1/2 by OSI-027 potently inhibits keloid keratinocyte proliferation and migration. www.impactjournals.com/oncotarget/ Vol.9, (No.1), Supplement 1, pp: s147-s154


INTRODUCTION
Keloid is characterized by the formation of exuberant and autonomous scar tissue that extends the clinical border of the original wound [1][2][3].It often forms as a result of dermal injuries, and is considered, to a limited extent, as a benign tumor [1][2][3].A set of different chemokines, cytokines and growth factors are oversecreted, which stimulate the proliferation and migration of keratinocytes (and other skin cells) to form keloid skin [1][2][3].The hyper-activation of the serine/threonine protein kinase mammalian target of rapamycin (mTOR) plays a pivotal role in the process [4][5][6][7][8].
In the current study, we found that targeting mTORC1/2 withOSI-027 inhibited the proliferation and migration of keloid keratinocytes, where mTOR was found to be overactivated.
As described, primary human keratinocytes derived from keloid skin and surrounding normal skin were established and cultured.Viable cell counting assay results demonstrated that the keloid keratinocytes grew faster than the normal keratinocytes (Figure 1A).Meanwhile, an MTT OD assay further showed increased proliferation of keloid keratinocytes compared to normal keratinocytes (Figure 1B).Moreover, in keloid keratinocytes, BrdU (Figure 1C) and [H 3 ] DNA incorporation (Figure 1D) were both increased.These results demonstrate the increased cell proliferation of keloid primary keratinocytes compared to normal keratinocytes (Figure 1A-1D).
Migration of the above-mentioned cells was also tested using phagokinetic track motility [4,21] and transwell assays.The former measured the migration distance of each single keratinocyte [4,21], while the latter analyzed the number of migrated cells.As compared to normal keratinocytes, both migration distance (Figure 1E, quantified results are shown in the right panel) and the number of migrated cells (Figure 1F) were increased for keloid keratinocytes.Collectively, these results demonstrate enhanced cell proliferation and migration in keloid keratinocytes.

mTORC1 and mTORC2 are both important in proliferation and migration of keloid keratinocytes
Next, constitutivelyactiveS6K1 (T389E, "ca-S6K1flag-puro") [24] was introduced to keloid keratinocytes.As shown in Figure 4A, the introduction of ca-S6K1 completely restored S6K1 phosphorylation in OSI-027treated keratinocytes.S6K1 phosphorylation was even higher than the control level (Figure 4A, quantification).The activation of mTORC2, detected by anti-p-AKT at Ser-473, was still blocked after OSI-027 treatment in ca-S6K1-expressing cells (Figure 4A).Intriguingly, inhibition of the OSI-027-induced proliferation (BrdU ELISA assay, Figure 4B) of keloid keratinocytes was only partly inhibited by ca-S6K1.Similarly, Figure 4C demonstrates that ca-S6K1 only partly restored the migration potentialofOSI-027-treated cells as determined by a phagokinetic track migration assay.It should be noted that the mTORC1 blocker, rapamycin (also at 100 nM) was less potent than OSI-027 in inhibiting the proliferation and migration of keloid keratinocytes (Figure 4B and 4C).These results imply that the inhibition of mTORC1 less potently inhibited keloid keratinocytes than OSI-027, suggesting that activation of both mTORC1 and mTORC2 is required in the process.
To further confirm our hypothesis, an siRNA strategy was applied to silence Rictor, which is the key component of mTORC2 [15].In keloid keratinocytes, the two non-overlapping Rictor siRNAs ("Rictor siRNA I/II") both induced dramatic knockdown of Rictor (Figure 4D).Consequently, phosphorylation of the mTORC2 substrate, AKT (at Ser-473), but not the mTORC1 substrate, S6K1, was largely inhibited (Figure 4D).Notably, the silencing of Rictor by targeted siRNAs also inhibited proliferation (Figure 4E) and migration (Figure 4F) of keloid keratinocytes.However, Rictor siRNA-mediated inhibition of keloid keratinocytes was less potent than OSI-027 (Figure 4B-4F).These results further support the notion that mTORC1 and mTORC2 are both important for the proliferation and migration of keloid keratinocytes.

DISCUSSION
A skin keloid is characterized by the hyper-activity of keratinocytes (and other skin cells) within the wound [1][2][3].
Due to these limitations, direct mTOR kinase inhibitors have recently been developed, termed "secondgeneration mTOR inhibitors" [26].OSI-027 is a novel  E and F).For all assays, the same exact number of viable keratinocytes from keloid or surrounding normal skin was initially plated into each well (same for all figures).Data are presented as mean ± standard deviation (SD).* p < 0.05 vs. "normal skin" keratinocytes.Bar = 25 μm (E).Experiments were repeated three times, and similar results obtained.
We, here, propose that both mTORC1 and mTORC2 are important in promoting the proliferation and migration of keloid keratinocytes for the following reasons: First, mTORC2, similar to mTORC1, is over-activated in keloid keratinocytes.Second, the mTORC2 substrate AKT (phosphorylation at Ser-473) is known to stimulate cell proliferation and migration [16,17,32].Third, the cocurrent blockage of mTORC1/2 by OSI-027 was more potent than rapamycin (mTORC1 inhibitor) in inhibiting keloid keratinocytes.Additionally, restoring mTORC1 activation by ca-S6K1 only partly attenuated OSI-027-induced inhibition of keloid keratinocytes.Lastly, mTOR2 inhibition, by Rictor siRNAs, inhibited keloid keratinocyte proliferation and migration, less efficiently than OSI-027.Therefore, these in vitro results imply that targeting both mTORC1 and mTORC2 by OSI-027 may have translational value in treating keloid skin.

Culture of primary human keratinocytes
Epidermal tissues of keloid skin and surrounding normal skin were obtained via biopsy from five informconsent keloid patients (Male, 29/32/35/41/47-year old, administrated at Shanghai Ninth People's Hospital, Shanghai, China).The patients received no prior treatment.The acquired epidermal tissues were minced, washed, and digested by collagenase I (Sigma-Aldrich, catalog no.C0130) [34].Digestions (2-5) were neutralized, pooled, and filtered.Primary cultures of human keratinocytes were then maintained in Medium 154-CF (Cascade Biologics, M154CF, Portland, OR, USA) supplemented with Human Keratinocyte Growth Supplement plus antibiotics and Ca 2+ (catalog no.S0015) [4].The medium was provided by Dr. Cao [4].Skin fibroblasts were abandoned.Protocols using human tissues and cells were conducted according to the principles of the Declaration of Helsinki, and were approved by the Ethics Review Board (ERB) of all authors' institutions.Written, informed consents were obtained from the participants.

BrdU DNA incorporation assay
Primary human keratinocytes were seeded into 48-well tissue culture plates (5 × 10 4 cells/well) and cultured for 72 h.A 5-bromo-2′-deoxyuridine (BrdU) dye was then added for an additional 16 h.BrdU incorporation into DNA was tested via a BrdU ELISA colorimetric assay (Roche, Shanghai, China, catalog no.11647229001) according to the manufacturer's protocol.The ELISA OD value at 450 nM was recorded.

[H 3 ] Thymidine DNA incorporation assay
Primary human keratinocytes were seeded into 48well plates (5 × 10 4 cells/well) and cultured for 72 h in the presence of 1 μCi/mL of tritiated thymidine (Sigma-Aldrich).To determine [H 3 ] thymidine incorporation, cells were washed with cold PBS, and DNA precipitated and solubilized as described previously [4].DNA aliquots were counted and quantified by liquid-scintillation spectrometry, and the [H 3 ] thymidine DNA OD recorded.

Phagokinetic track migration assay
A detailed protocol has been previously described [21,35,36].Briefly, 12-well tissue culture plates were coated with fibronectin (10 μg/mL, Sigma-Aldrich, catalog no.10838039001), and wells then washed with PBS.Afterwards, a microsphere (Sigma-Aldrich, catalog no.NIST1965) suspension was then added to each well.The plates were then centrifuged at 500 × g at 4°C for 20 min.After the supernatant was removed, and freshly trypsinized keratinocytes (1000 cells per well of each treatment) seeded for 48 h, cell migration was observed and photographed under a light microscope.

Transwell assay
The transwell chamber with 12 μm pore filters (Corning, 3403, Shanghai, China) was first pre-coated with 1 mg/mL Matrigel (BD Biosciences, catalog no.354230, Suzhou, China).Afterwards, keratinocytes (3 × 10 4 cells per chamber) in serum-free medium were plated into the upper chamber.Complete medium (with fetal bovine serum [FBS]) was added to the lower chamber.After 48 h of incubation, cells on the lower surface of the membrane were fixed, stained and then counted in five random fields [37].Mitomycin (1.5 μg/mL; Sigma-Aldrich) was always added to exclude the influence of cell proliferation.

Propidium iodide assay for cell death
After treatment, keratinocytes were washed with icecold PBS, and re-suspended in 70% alcohol, followed by the addition of DNase-free RNase and propidium iodide (PI, Sigma-Aldrich, catalog no.P4170).The suspension was filtered and subjected to analysis by flow cytometer (FACSCalibur; BD Biosciences).The PI percentage was recorded.

Western blotting assay
Keratinocytes were lysed via radioimmunoprecipitation assay (RIPA) lysis buffer (Biyuntian, Wuxi, China).Thirty µg of protein lysate per treatment was separated by a SDSpolyacrylamide gel electrophoresis (PAGE) gel, which was then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, catalog no.IPVH00010, Shanghai, China).After blocking in 5% of milk, the blot was incubated with indicated primary and corresponding secondary antibodies.Enhanced chemiluminescence (ECL) reagents (Amersham Biosciences, Shanghai, China) were applied to detect antibody-antigen binding.The total gray of each band was quantified via Image J software (NIH, Bethesda, MD).Each lane was loaded with the same amount of quantified protein lysate.The same set of lysate samples were run in sister gels to test different proteins.

Co-immunoprecipitation
The co-immunoprecipitation (Co-IP) assay protocol has been described previously [38].Briefly, for each treatment, 800 μg protein lysates were pre-cleared with protein A/G beads (Sigma, catalog no.11719416001) and then incubated with anti-mTOR antibody (0.2 μg), overnight.Protein A/G beads (30 μL per treatment, Sigma) were added afterwards to the lysates again for another 4 h.The beads were then washed with PBS five times, and one time with cell lysis buffer.The integrity of the mTOR complex was then detected by western blotting assay.

Rictor siRNA
Keloid keratinocytes were seeded into a six-well tissue culture plate at 1 × 10 5 cells per well, grown for 24 h, and transfected with Rictor siRNA I (Cell Signaling Technology, #8649), Rictor siRNA II (Cell Signaling Tech, #8622), or scramble control siRNA (Cell Signaling Technology, #6568), at a final concentration of 100 nM using Lipofectamine 2000 reagent (Invitrogen).To exam siRNA efficiency, cells were harvested 48 h after siRNA transfection, and the silencing of Rictor in transfected cells was confirmed by western blot assay.

Statistics
Data were expressed as mean ± standard deviation (SD).The differential significance was calculated using ANOVA (SPSS 16.0).A p value of less than 0.05 was considered statistically significant.

Figure 1 :
Figure 1: Enhanced cell proliferation and migration in keloid keratinocytes.Primary cultured human keratinocytes, derived from keloid and surrounding normal skin, were subjected to cell counting (A), MTT (B), BrdU ELISA (C) and [H 3 ] DNA incorporation assays (D) to determine cell proliferation.Cell migration was analyzed using the Phagokinetic track migration assay listed in the text (E and F).For all assays, the same exact number of viable keratinocytes from keloid or surrounding normal skin was initially plated into each well (same for all figures).Data are presented as mean ± standard deviation (SD).* p < 0.05 vs. "normal skin" keratinocytes.Bar = 25 μm (E).Experiments were repeated three times, and similar results obtained.

Figure 2 :
Figure 2: OSI-027 potently inhibits proliferation and migration of keloid keratinocytes.Primary cultured human keratinocytes, derived from keloid and surrounding normal skin, were subjected to a western blotting assay of listed proteins (A, data of four repeated blots were quantified).Primary keloid keratinocytes were treated with OSI-027 (100 nM), vehicle control (0.1% of DMSO, "C") or H 2 O 2 (300 μM).Cell proliferation (B-D, 72 h), cell migration (E and F, 48 h) and cell death (G, 72 h) were tested by the assays listed in the main text.Data are presented as mean ± standard deviation (SD).* p < 0.05 vs. "normal skin" keratinocytes (A) or "C" group (B-G).Bar = 25 μm (E).Experiments were repeated three times, and similar results obtained.

Figure 3 :
Figure 3: OSI-027 blocks mTORC1 and mTORC2 activation in keloid keratinocytes.Cultured primary keloid keratinocytes were treated with vehicle control (0.1% of DMSO, "C") or OSI-027 (100 nM) for 2 h.Co-immunoprecipitation (Co-IP) was performed to test the association between mTOR and mTOR complex proteins (Rictor, Raptor and mLST8) (A, left panel).The expression of listed proteins was examined by western blot (A, inputs, and B-D).Experiments were repeated three times, and similar results obtained.

Figure 4 :
Figure 4: The importance of mTORC1 and mTORC2 in the proliferation and migration of keloid keratinocytes.