Abstract

   Cancer cells frequently exhibit aberrant redox homeostasis and
   adaptation to oxidative stress. Hence abrogation of redox adaptation in
   cancer cells can be exploited for therapeutic benefit. Here we report
   SGK3 functions as an anti-oxidative factor to promote cell growth and
   drug resistance in cervical cancers harboring PIK3CA helical domain
   mutations. Mechanistically, SGK3 is activated upon oxidative stress and
   exerts anti-ROS activity by stabilizing and activating the antioxidant
   enzyme catalase. SGK3 interacts with and phosphorylates catalase,
   promoting its tetrameric state and activity. Meanwhile, SGK3
   phosphorylates GSK3β and protects catalase from GSK3β-β-TrCP mediated
   ubiquitination and proteasomal degradation. Furthermore, SGK3
   inhibition not only potentiates CDK4/6 inhibitor Palbociclib-mediated
   cytotoxicity, but also overcomes cisplatin resistance through
   ROS-mediated mechanisms. These data uncover the role of SGK3 in
   maintaining redox homeostasis and suggest that the SGK3-catalase
   antioxidant signaling axis may be therapeutically targeted to improve
   treatment efficacy for cervical cancers carrying PIK3CA helical domain
   mutations.

   Keywords: SGK3, Catalase, PIK3CA, Cervical cancer, Redox homeostasis,
   Antioxidative

Graphical abstract

   [37]Image 1
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Abbreviations

   3D
          three dimensional

   8-OHdG
          8-hydroxydeoxyguanosine

   CAT
          catalase

   CCK-8
          cell counting kit-8

   CHX
          cycloheximide

   DCFH-DA
          2′,7′-dichlorofluorescin diacetate

   DHE
          dihydroethidium

   DOX
          doxycycline

   EnR
          endoplasmic reticulum

   ER
          estrogen receptor

   GSEA
          gene set enrichment analysis

   HPV
          human papillomavirus

   hVPS34
          class III PI3K

   INPP4B
          Inositol polyphosphate 4-phosphatase type II

   KD
          knockdown

   MS
          mass spectrometry

   NAC
          N-Acetyl-l-cysteine

   OD
          optical density

   PD
          palbociclib

   PI3K
          phosphatidylinositol 3 kinase

   Rb
          retinoblastoma protein

   ROS
          reactive oxygen species

   RPPA
          reverse phase protein array

   SERCA
          sarcoplasmic/EnR calcium ATPase 2b

   SGKs
          serum and glucocorticoid-inducible protein kinases

   SILAC
          stable isotope labeling of amino acids

   STR
          short tandem repeat

   TCGA
          the cancer genome atlas

1. Introduction

   Cervical cancer remains one of the most common causes of cancer-related
   death among women worldwide [[39]1]. Although early-stage cervical
   cancer can be treated with surgery or radiotherapy to prolong survival,
   patients with advanced, recurrent or metastatic cervical cancer have
   very limited treatment options after receiving platinum-based
   chemotherapy [[40]2]. There is therefore an urgent need to develop
   effective therapeutic strategies to improve treatment efficiency for
   cervical cancer and overcome therapy resistance.

   Hyperactivation of the phosphatidylinositol 3 kinase (PI3K) signaling
   pathway occurs frequently in a variety of human malignancies including
   cervical cancer [[41][3], [42][4], [43][5]]. As most of the studies
   have focused on AKTs as the effectors that transduce signals from PI3Ks
   to malignant phenotypes, a large number of PI3K pathway inhibitors
   including pan-/isoform-specific PI3K inhibitors and AKT inhibitors have
   entered clinical development for cancer treatment [[44]3,[45]6].
   However, recent studies point to SGK3 as a critical effector of
   oncogenic PIK3CA mutant cancers that are independent of AKT [[46][7],
   [47][8], [48][9]]. Serum and glucocorticoid-inducible protein kinases
   (SGKs) including SGK1, SGK2 and SGK3 are members of the AGC family of
   serine/threonine kinases and effectors downstream of Class I PI3Ks
   [[49]10,[50]11]. SGK isoforms share more than 50% sequence identity
   with the kinase domains of AKT isoforms [[51]12,[52]13]. Unlike AKT
   isoforms or the other two SGK family members (SGK1 and SGK2), SGK3
   possesses a unique N-terminal PX domain which binds PtdIns-3-P. The
   PtdIns-3-P binding has been shown as a prerequisite for phosphorylation
   at Thr320 by PDK1 and at Ser486 by mTORC2, leading to full activation
   of SGK3 [[53][9], [54][10], [55][11]]. As PtdIns-3-P can be generated
   by Class III PI3K/hVPS34 at the endosome or inositol polyphosphate
   4-phosphatase B (INPP4B) on the membrane, it has been proposed that
   SGK3 activation induced by binding to PtdIns-3-P is controlled by VPS34
   and INPP4B [[56]10,[57]14,[58]15]. Aberrant activation of SGK3 has been
   reported to contribute to tumorigenesis and/or drug resistance in a
   variety of cancer types including breast, liver, and prostate cancers
   [[59]7,[60]8,[61]16,[62]17]. Several lines of evidence indicate that
   tumors with the PIK3CA H1047R mutation are predominantly dependent on
   AKT activation, whereas tumors (particularly cervical cancer) with the
   PIK3CA E542/E545K mutation more often exhibit low AKT activity and
   selectively rely on SGK3 kinase activity for survival
   [[63]7,[64]8,[65]18,[66]19]. However, the precise oncogenic role of
   SGK3 in cervical cancer related to the setting of PI3K helical domain
   mutations remains obscure.

   Almost all cervical cancers (99%) are caused by chronic infection with
   oncogenic human papillomavirus (HPV) [[67]4,[68]5]. Reverse phase
   protein array (RPPA) analysis of HPV-positive clinical specimens
   harboring activating PIK3CA mutations demonstrated robust mTOR
   signaling but the lack of AKT activation [[69]20], suggesting the
   existence of AKT-independent effector signaling downstream of PI3K with
   HPV positivity. Persistent HPV infection often leads to enhanced
   cellular oxidative stress [[70]21]. To mitigate oxidative stress,
   cancer cells have developed an enhanced antioxidant capacity to survive
   and confer drug resistance [[71]22,[72]23]. Accumulating evidence
   indicates that upregulated oxidative stress response by activation of
   ROS scavenging system may play important roles in the pathogenesis of
   cervical cancer [[73]24]. In this scenario, cancer cells under high
   levels of oxidative stress are more susceptible to damage due to
   abrogation of redox adaptation [[74]25]. Therefore, understanding the
   redox regulatory mechanisms by which cervical cancer cells maintain
   redox homeostasis is important for the development of new anti-cancer
   strategies. Our recent work shows that SGK1 exerts anti-ROS activity in
   cervical cancer cells by upregulating the expression and activity of
   NRF2 [[75]26], the master regulator of antioxidant responses. However,
   whether SGK3 is also involved in regulation of ROS homeostasis in
   cervical cancer has not been reported.

2. Results

2.1. Activation of SGK3 augments malignant behavior of cervical cancer cells

   The PIK3CA mutations frequently found in human cancers are mainly
   localized to the helical domain and kinase domain of the PI3K catalytic
   subunit p110α [[76]3]. Notably, analysis of The Cancer Genome Atlas
   (TCGA) data showed that the helical domain mutation E542/545K
   predominated in cervical cancer whereas the kinase domain mutation
   H1047R occurred at a much lower frequency ([77]Fig. 1A). Such
   distribution pattern in cervical cancer was distinct from that in other
   women's malignancies including breast, ovarian and endometrial cancers
   ([78]Fig. 1A). Further analysis of TCGA reverse phase protein array
   (RPPA) data revealed that tumors carrying PIK3CA H1047R were
   significantly associated with AKT activation as determined by the
   levels of phospho-AKT (p-AKT^S473 and p-AKT^T308) ([79]Fig. 1B). In
   contrast, tumors carrying PIK3CA wild-type (WT), PIK3CA E542/545K or
   other PIK3CA mutations, but not PIK3CA H1047R exhibited comparable
   p-AKT levels. These observations support the notion that PIK3CA
   E542/545K may promote cervical cancer growth through an AKT-independent
   mechanism [[80]7,[81]18,[82]19]. SGK3 is thought to be the primary
   downstream effector that transduces the oncogenic signaling from PIK3CA
   E542/545K [[83]7,[84]8,[85]18]. In line with this, we observed a
   correlation between PIK3CA E545K and SGK3 activation as evidenced by
   the levels of phospho-SGK3 (p-SGK3^T320) in a panel of human cervical
   cancer cell lines ([86]Fig. 1C). Interestingly, while treatment with
   SGK3-PROTAC1 [[87]27], a small molecule degrader of SGK3 protein,
   significantly attenuated the growth of ME180 and CaSki cells (both
   PIK3CA E545K), it had only a moderate effect on the growth of SiHa and
   HT-3 cells (both PIK3CA WT) ([88]Figs. 1D and S1A). These data suggest
   that cervical cancer cells with the PIK3CA E545K mutation may depend on
   SGK3 for survival.

Fig. 1.

   [89]Fig. 1
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   Activation of SGK3 promotes malignant growth of cervical cancer cells.
   (A) The percentages of E542/545K, H1047R and other PIK3CA mutant tumors
   in breast, cervical, ovarian and endometrial cancers from TCGA are
   shown. ***P < 0.001(Chi-square test). (B) Reverse phase protein array
   (RPPA) analysis of p-AKT^S473 and p-AKT^T308 levels (Z-scores) in the
   tumor samples as specified in “materials and methods”. PIK3CA WT,
   n = 657; E542/545K, n = 112; H1047R, n = 103; Other mutations, n = 126.
   *P < 0.05, ***P < 0.001 (one-way ANOVA with Tukey's multiple
   comparisons test). (C) Western blot showing expression of p-SGK3^T320
   and SGK3 in cervical cancer cell lines as indicated. (D) Cervical
   cancer cells were treated with or without SGK3-PROTAC1 to measure cell
   viability. (E) SiHa cells with ectopic overexpression of SGK3 wild-type
   (WT), S486D, K191 M or Vector were cultured in three-dimensional (3D)
   Matrigel. Representative images are shown. Scale bars, 100 μm. (F)
   Western blot analysis of the indicated proteins in cells cultured as
   described in (E). (G) Tumor growth curves of mice bearing xenografted
   tumors derived from SiHa cells stably expressing SGK3 WT, S486D, K191 M
   or Vector. n = 6–8 tumors, per group. Data are shown as Mean ± S.E.M.
   ***P < 0.001 (two-way ANOVA with Bonferroni's Tukey's multiple
   comparisons test). ns, not significant, *P < 0.05, ***P < 0.001. (C and
   F)) Vinculin was used as a loading control. (C–F) Data are shown as
   Mean ± S.D. for three independent experiments. ns, not significant,
   *P < 0.05, **P < 0.01, ***P < 0.001 (two-way ANOVA with Bonferroni's
   Tukey's multiple comparisons test).

   Emerging evidence suggests that SGK3 mediates important cellular
   processes involved in malignant transformation [[91]9,[92]13,[93]14].
   To assess the role of SGK3 kinase activity in cervical tumorigenesis,
   we stably expressed WT (wild-type), S486D (constitutively active) or
   K191 M (kinase-dead)-mutant SGK3 in SiHa cells ([94]Fig. S1B) [[95]28].
   Strikingly, while ectopic expression of SGK3 WT or S486D resulted in
   significantly enhanced cell growth and migration potential, SGK3 K191 M
   exhibited moderate inhibitory effects, indicating the oncogenic
   potential of SGK3 activation in cervical cancer ([96]Fig. S1C and S1D).
   When cultured under three dimensional (3D) conditions, the
   constitutively active mutant SGK3 S486D-expressing SiHa cells grew more
   robustly than the SGK3 WT-expressing cells ([97]Fig. 1E). This
   observation was correlated with SGK3 activation as evidenced by
   substantially increased levels of p-SGK3 and its downstream effectors
   including p-GSK3β, p-S6RP and p-4EBP1 ([98]Fig. 1F). In contrast, SiHa
   cells expressing the kinase-dead mutant SGK3 K191 M exhibited
   diminished SGK3 signaling and moderate growth in 3D Matrigel ([99]Fig.
   1E and F). We next tested the impact of SGK3 activation on cervical
   tumorigenesis by establishing SiHa cell-derived xenografts in
   immunodeficient mice. Indeed, ectopic expression of SGK3 WT and, to a
   dramatic extent, SGK3 S486D accelerated tumor formation and growth
   ([100]Figs. 1G and S1E), whereas ectopic expression of SGK3 K191 M
   markedly reduced tumor burden compared to the control, suggesting that
   SGK3 activation promotes cervical tumor growth.

2.2. SGK3 promotes CAT stabilization and activation through direct
phosphorylation of CAT at Ser167

   To gain insights into the potential biological consequences of SGK3
   activation, we assessed the impact of the constitutively active SGK3
   mutant S486D on the proteome in SiHa cervical cancer cells by stable
   isotope labeling of amino acids (SILAC)-based mass spectrometry (MS)
   analysis ([101]Fig. 2A). Enrichment-based clustering analysis of KEGG
   pathway, Gene Ontology (biological process and molecular function)
   revealed that the upregulated proteins and pathways in SiHa cells with
   SGK3 activation are highly enriched in oxidative phosphorylation and
   reactive oxygen species (ROS) ([102]Figs. 2B, S2A and S2B). Meanwhile,
   to identify proteins associated with the catalytically active form of
   SGK3 protein (S486D), we performed immunoprecipitation with an SGK3
   antibody followed by SILAC-MS analysis. Consistent with a previous
   report [[103]29], we observed the association of SGK3 with heat shock
   protein 90α (encoded by HSP90AA1) ([104]Fig. 2C). Among the other
   identified binding partners of the SGK3 S486D protein, the hydrogen
   peroxidase Catalase (CAT) caught our attention as a potential substrate
   of SGK3 due to the fact that CAT is an antioxidant enzyme and that CAT
   can be phosphorylated and activated by PKCδ [[105]30], also an AGC
   kinase family member.

Fig. 2.

   [106]Fig. 2
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   SGK3 mediates CAT function through phosphorylation of CAT at Ser167.
   (A) Schematic of SILAC-based experiments analyzed in this study (see
   Methods for more details). (B) KEGG pathway enrichment analysis of the
   proteome in SiHa cells with ectopic overexpression of SGK3 S486D (the
   catalytically active mutant). SGK3 S486D, Light (L); Vector, Heavy (H).
   All the quantified proteins were divided into four quantiles (Q1 ∼ 4)
   according to L/H ratios: Q1 (Ratio ≤ 0.67), Q2 (0.67 < Ratio < 0.77),
   Q3 (1.3 < Ratio < 1.5), Q4 (Ratio ≥ 1.5). (C) The identity of SGK3
   S486D protein-binding partners uncovered by mass spectrometry analysis.
   The color intensity is shown as the fold change of protein abundance of
   SGK3 S486D binding partners in SGK3 S486D-expressing versus
   Vector-expressing SiHa cells. (D) The interaction between endogenous
   CAT and ectopically expressed SGK3 was examined by immunoprecipitation
   (IP) using an anti-Flag antibody, followed by Western blot analysis in
   SiHa cells stably expressing Vector, SGK3 WT or S486D. (E)
   Immunofluorescence analysis of CAT (green) and Flag-SGK3 S486D (red) in
   SiHa cells stably expressing SGK3 S486D. Scale bars: 25 μm. (F)
   Sequences containing CAT Ser167 are highly conserved across species as
   indicated. (G) Extracts of CaSki cells stably expressing CAT WT, S167A
   or S167D were subjected to crosslinking by 0.025 wt% glutaraldehyde,
   followed by Western blot analysis for CAT protein levels. Tetrameric,
   dimeric, and monomeric CAT proteins were indicated. (H) Extracts of
   SiHa cells stably expressing Vector, SGK3 WT, S486D or K191 M were
   subjected to crosslinking by 0.025 wt% glutaraldehyde, followed by
   Western blot analysis for CAT protein levels. (I) SiHa cells stably
   expressing SGK3 S486D or K191 M were treated with 20 μg/ml
   cycloheximide (CHX) over a time course, followed by Western blot
   analysis for CAT expression. (D, G and H) Vinculin was used as a
   loading control. (D, G-I) Data are shown as Mean ± S.D. for three
   independent experiments. ns, not significant, ***P < 0.001 (two-way
   ANOVA with Bonferroni's Tukey's multiple comparisons test). (For
   interpretation of the references to color in this figure legend, the