Abstract

   Small molecule inhibitors of apoptosis proteins (IAPs) antagonists,
   known as Smac mimetics (SMs), activate non-canonical NF-κB and
   sensitize cancer cells to TNF-induced cell death. SMs are currently in
   phase III clinical trials for head and neck squamous cell carcinoma
   (HNSCC) after promising phase II trials. To explore the utility of SMs
   in oral squamous cell carcinoma (OSCC), we tested nine human OSCC cell
   lines and correlated SM sensitivity with both IAP mutation and
   expression levels. cIAP1 protein expression was shown to be higher in
   OSCC and a predictor of poor prognosis. However, our in vitro and in
   vivo testing demonstrated differential sensitivity to SMs, which did
   not correlate with cIAP1 and cIAP2 expression in these OSCC cell lines.
   Exogenous TNF failed to effectively increase the sensitivity of
   SM-resistant OSCC cells to SM-induced cell death. SM resistance was
   associated with a deficiency in Complex IIa formation, but activation
   of non-canonical NF-κB was not a determinant of SM efficacy. Finally,
   metabolic analysis revealed that the ABC transporter pathway was
   activated in SM-resistant OSSC cells, and SMs combined with ABC
   transporter inhibitors improved cell death sensitivity to overcome SM
   resistance. These studies highlight the therapeutic potential of SMs in
   OSCC and support patient stratification to improve efficacy with the
   addition of adjuvant therapy.

   Subject terms: Cancer, Diseases

Introduction

   The most common sub-group of head and neck squamous cell carcinoma
   (HNSCC) and the most commonly diagnosed malignancy of the oral cavity
   is oral squamous cell carcinoma (OSCC) [[58]1, [59]2]. OSCC is a global
   health problem, with an increasing incidence in recent years [[60]3,
   [61]4] and a mortality rate that continues to rise, particularly in the
   older female population (over 65 years) [[62]5]. The standard of care
   for locally advanced OSCC and recurrent/metastatic OSCC is surgery
   combined with chemo-radiotherapy [[63]6], but the 5-year overall
   survival rate has not improved for decades [[64]7]. In addition,
   monoclonal antibodies, such as cetuximab (epidermal growth factor
   receptor inhibitor) have improved advanced HNSCC prognosis when
   combined with radiotherapy [[65]8], but this combinative therapy was
   less effective than traditional chemoradiation [[66]9]. First-line
   pembrolizumab (anti-PD-1 immune checkpoint inhibitor) with/without
   chemotherapy prolongs survival in populations with recurrent/metastatic
   HNSCC [[67]10]. However, the high rate of adverse events (53.3%)
   [[68]11] and failure to significantly improve efficacy [[69]12] were
   reported in OSCC clinical trials. Therefore, there is a pressing need
   to explore new therapeutics for OSCC.

   SMAC mimetics (SMs) are small peptide-like molecules, that mimic the
   action of the SMAC protein, the endogenous antagonist of inhibitors of
   apoptosis proteins (IAPs) [[70]13]. Since cIAPs play an important role
   in activating the TNF-associated survival pathway, their antagonists
   sensitize cells to TNF-induced cell death [[71]14, [72]15]. Therefore,
   the anti-cancer activity of SMs is closely correlated to TNF signaling.
   Apart from TNF-mediated extrinsic apoptosis, SMs also potentially
   sensitize cells to the intrinsic apoptotic pathway. Upon receipt of an
   intrinsic apoptotic signal, Bax and Bak oligomerize in the outer
   mitochondrial wall to form a pore that permits SMAC to enter the
   cytosol and bind to and inhibit IAPs, particularly XIAP, the caspase-3
   caspase-9 inhibitor [[73]16, [74]17]. Nevertheless, HNSCC is associated
   with mutations in genes such as cIAP1 [[75]18]/BIRC2 [[76]19],
   caspase-8 [[77]20], FADD [[78]19], and TNF [[79]21] that affect the
   extrinsic apoptosis pathway indicating that this is pathway is likely
   to be particularly relevant when exploring SM induced cell death in
   HNSCC.

   Due to the good safety profile and promising anti-tumor effects,
   especially in certain murine transplantation models [[80]22–[81]24],
   multiple clinical trials of SMs are underway. To date, SMs including
   GDC-0152, GDC-0917, AT406, LCL161, BI 891065, birinapant, APG-1387,
   ASTX660, and HGS1029 have been evaluated in clinical trials for various
   solid and hematological cancers [[82]13, [83]25]. However, as single
   agents, SMs rarely induce complete responses in patients and a number
   of clinical trials have been terminated due to lack efficacy [[84]26,
   [85]27]. However, as adjuvants to improve sensitivity to radiotherapy,
   chemotherapy, and immunotherapy, SMs are proving to be more beneficial
   [[86]13]. Therefore, defining the mechanisms of impaired anti-tumor
   activities in cancer cells, resulting in clinical trial failure is
   urgently required.

   Notably, SM-induced cell death also appears to be dependent on the
   tumor molecular subtype. For example, our previous research had
   demonstrated that SMs were more effective in triple-negative breast
   cancers than in other subtypes [[87]28]. Moreover, in colorectal
   cancers, SMs had shown to increase the sensitivity of Consensus
   Molecular Subtypes 4 (CMS4) cells to oxaliplatin/5-FU, whereas CMS2
   cells were unresponsive [[88]29, [89]30]. This indicated that the
   molecular classification of patients may enhance the efficacy of SMs in
   clinical trials.

   Clinical trials with SMs, including AT406, birinapant, APG-1387, and
   ASTX660 have been conducted in HNSCC, where safety was proven in phase
   I trials [[90]31]. Of these, AT406 has commenced recruiting for HNSCC
   phase III clinical trials ([91]NCT04459715, [92]NCT05386550), since the
   encouraging results of the Phase II trial [[93]32, [94]33].
   Unfortunately, the latter trial included very few OSSC patients.
   Therefore, it is imperative to combine the clinicopathological
   characteristics of OSCC patients with molecular factors that may confer
   therapy sensitivity or resistance to assess the future feasibility of
   SM treatment for OSCC.

   In order to define markers of SM sensitivity, this study assessed the
   expression of SM targets, namely cIAP1 and cIAP2, in OSCC cell lines
   and patients. Furthermore, this study compared the expression levels of
   key proteins and metabolic profiles in SM-sensitive and SM-resistant
   cells. While several known SM sensitivity factors, such as
   non-canonical NF-κB, did not correlate with sensitivity, we found that
   elevated levels of ABC family members (ABCA3, ABCB1, and ABCB4) and
   failure of Complex IIa formation contributed to SM resistance in OSCC.

Results

cIAP1 is aberrantly expressed in OSCC and predicts poor clinical outcome

   Since SM-induced cell death relies on the ability to antagonize IAPs,
   we first evaluated DNA mutation, mRNA, and protein expression of BIRC2
   (encoding cIAP1) and BIRC3 (encoding cIAP2) in the TCGA database
   [[95]34, [96]35] and from our OSCC clinical sample bio-bank (Affiliated
   Stomatological Hospital, Medical School of Nanjing University) to
   determine whether the targets of SMs were aberrantly expressed in OSCC.
   Most of SMs such as birinapant [[97]36], AT406 [[98]37], LCL161
   [[99]38], ASTX660 [[100]39], and AZD5582 [[101]40] have higher affinity
   for cIAP1 and/or cIAP2 than XIAP, therefore we excluded XIAP expression
   analysis in this study. In the TCGA, PanCancer Atlas [[102]34], we
   found that 5.35% and 5.74% of HNSCC patients had BIRC2 or BIRC3
   amplification mutations respectively (Fig. [103]1A). In an additional
   pan-cancer dataset (ICGC, TCGA, Pan-cancer whole genome analysis,
   Nature 2020) [[104]35], we found that HNSCC patients had the second
   highest BIRC2 and BIRC3 mutation frequency among 27 types of cancers
   with a prevalence of 14.29% (Supplementary Fig. [105]1A), similar to
   Eytan et al. [[106]19]. To specifically obtain the mutation landscape
   in OSCC patients, we further analyzed the BIRC2 and BIRC3 amplification
   mutation rates in our OSCC bio-bank samples. We found that the
   amplification mutation of BIRC2 and BIRC3 was 4.81% (9/187) and 6.42%
   (12/187) (Fig. [107]1B), which was similar to the proportions in HNSCC
   patients. These data suggested that amplification mutation of BIRC2 and
   BIRC3 exists in HNSCC patients, including OSCC patients.

Fig. 1. BIRC2 and BIRC3 mutations in OSCC and HNSCC patients.

   [108]Fig. 1
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   A Bar graph showing BIRC2 and BIRC3 DNA alterations in HNSCC patients,
   n = 523. Data from TCGA, PanCancer Atlas. B Bar graph showing BIRC2 and
   BIRC3 DNA amplification mutations in OSCC patients, n = 187. Patient
   DNA samples from OSCC Clinical Sample Bank of Nanjing Stomatological
   Hospital. C Comparison of the cIAP1 expression between OSCC patients
   with (n = 9)/without (n = 14) BIRC2 amplification mutation. P = t test.
   D Comparison of the cIAP2 expression between OSCC patients with
   (n = 12)/without (n = 14) BIRC3 amplification mutation. P = t test. E
   Representative IHC staining and graphical summary of cIAP1 in OSCC
   tissues from patients with (n = 9)/without (n = 14) BIRC2 amplification
   mutation. F Representative IHC staining and graphical summary of cIAP2
   in OSCC tissues from patients with (n = 12)/without (n = 14) BIRC3
   amplification mutation. Graphical depiction of overall survival (OS)
   (G), metastasis-free survival (MFS) (H), recurrence-free survival (RFS)
   (I), and disease-free survival (DFS) (J) from low transcription
   (n = 47) and high transcription mRNA levels (n = 41) of BIRC2 in OSCC
   patients, P = Log-rank (Mantel–Cox) test. Graphical depiction of OS
   (K), MFS (L), RFS (M), and DFS (N) from low transcription (n = 41) and
   high transcription mRNA levels (n = 47) of BIRC3 in OSCC patients,
   P = Log-rank (Mantel–Cox) test. Error bars are Mean ± SD.

   Moreover, to clarify the clinical relevance of the amplification
   mutations, we performed survival analysis of OSCC patients with/without
   BIRC2/BIRC3 amplification mutation. In comparison to the survival
   curves of normal OSCC patients, patients bearing BIRC2/BIRC3
   amplification mutation had a tendency to have a worse prognosis
   (Supplementary Fig. [110]2A–D). But the number of cases involved was
   insufficient to achieve statistical significance. Furthermore, we
   collected these patients’ frozen tumor tissues from our biobank to
   prepare paraffin sections for Immunohistochemistry (IHC) for
   cIAP1/cIAP2. We observed that OSCC patients with BIRC2/BIRC3
   amplification mutation exhibited higher expression levels of cIAP1
   (P = 0.0204) and cIAP2 (P = 0.0001) (Fig. [111]1C–F), which may be
   correlated to the poorer prognosis.

   BIRC2 and BIRC3 mRNA levels in tumor and paired non-tumor tissues in
   HNSCC patients were further analyzed. The data was extracted from the
   TCGA database [[112]34] and a comparative analysis revealed that BIRC2
   mRNA levels were significantly higher in tumor tissues compared to
   normal tissues (P = 0.0028) (Supplementary Fig. [113]2E). BIRC3 mRNA
   levels were not significantly different between tumor and normal
   tissues (Supplementary Fig. [114]2F). Subsequently, cDNA samples of 88
   OSCC patients were collected from our bio-bank and a prognostic value
   relating to BIRC2 and BIRC3 mRNA expression was analyzed. This analysis
   indicated that those with higher BIRC2 transcriptional levels had
   poorer overall survival (OS) (P = 0.0126), but it did not influence
   metastasis-free survival (MFS), recurrence-free survival (RFS) or
   disease-free survival (DFS) (Fig. [115]1G–J). Additionally, BIRC3 mRNA
   expression did not exhibit any significant influence on OS, MFS, RFS,
   and DFS (Fig. [116]1K–N). These findings suggest that a higher BIRC2
   transcriptional level contributed to OSCC progression.

   IHC staining for cIAP1 and cIAP2 proteins was conducted to examine the
   expression profile of these proteins in OSCC. Consistent with the mRNA
   analysis (Supplementary Fig. [117]2E), cIAP1 protein expression was
   higher in OSCC compared to normal oral epithelium (P < 0.0001) (Fig.
   [118]2A). However, despite the absence of significant differences in
   BIRC3 mRNA levels between tumor areas and normal epithelium in the TCGA
   database (P < 0.0001), high expression levels of cIAP2 were observed
   specifically in OSCC tumor areas compared to normal epithelium
   (P < 0.0001) (Fig. [119]2B, Supplementary Fig. [120]2F). Basing on the
   tumor tissue IHC score, patients were divided into low and high cIAP1
   /cIAP2 expression groups (Supplementary Fig. [121]3A, B). A chi-squared
   test was employed to analyze the diagnostic values of cIAP1 and cIAP2,
   which demonstrated that higher expression of cIAP1 was correlated with
   a higher mortality risk (P = 0.01), inferior differentiation
   (P = 0.038) and a higher worst pattern of invasion (WPOI) pathology
   grade (P = 0.001) (Supplementary Table [122]1).

Fig. 2. cIAP1 is aberrantly expressed in OSCC and predicts poor clinical
outcomes.

   [123]Fig. 2
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   Representative IHC staining and graphical summary of cIAP1 (n = 26) (A)
   and cIAP2 (n = 29) (B) in OSCC tissues and normal epithelium tissues
   from the same patient, P = paired t-test. C Representative images and
   graphical summary of cIAP1 expression in OSCC patients with WPOI I-III
   (n = 20) and WPOI IV-V (n = 42), P = t-test. D Representative images
   and graphical summary of cIAP2 expression in OSCC patients with WPOI
   I-III (n = 27) and WPOI IV-V (n = 44). P = t-test. Graphical depiction
   of OS (E), MFS (F), RFS (G), and DFS (H) for low (n = 31) and high
   expression levels (n = 31) for protein cIAP1, P = Log-rank (Mantel–Cox)
   test. Graphical depiction of OS (I), MFS (J), RFS (K), and DFS (L) for
   low (n = 36) and high expression levels (n = 35) for protein cIAP2,
   P = Log-rank (Mantel–Cox) test. Error bars are Mean ± SD.

   A positive correlation was observed between higher expression of cIAP2
   and poorer differentiation (P = 0.048) (Supplementary Table [125]2).
   Comparison of the expression levels of cIAP1 and cIAP2 in OSCC patients
   with WPOI I-III and WPOI IV-V revealed that patients with WPOI IV-V
   exhibited a higher expression of cIAP1 than those with WPOI IV-V
   (P = 0.0013) (Fig. [126]2C), whereas there was no significant
   difference in cIAP2 expression (Fig. [127]2D). In addition,
   Kaplan–Meier survival curve analysis indicated that patients with high
   cIAP1 expression levels exhibited reduced OS (P = 0.0077), RFS
   (P = 0.0426) and DFS (P = 0.0321) (Fig. [128]2E–H), while there was no
   significant correlation between cIAP2 expression and survival (Fig.
   [129]2I–L). Furthermore, multivariate Cox regression analysis adjusted
   for established clinical risk factors (eg. age at diagnosis, TNM stage,
   pathological stage, local recurrence, and distant metastasis) indicated
   that lymph node metastasis (HR = 3.31, P = 0.009), distant metastasis
   (HR = 2.802, P = 0.013) and high expression of cIAP1 (HR = 2.608,
   P = 0.035) were risk factors for OS in OSCC (Supplementary Table
   [130]3). Notably, high cIAP1 expression was identified as an
   independent prognostic factor for OSCC, which was not the case for
   cIAP2 (Supplementary Tables [131]3, [132]4). In conclusion, elevated
   cIAP1 expression was a predictor of a poor clinical outcome in OSCC.

OSCC tumor cells exhibit differential sensitivity to SM treatments

   Given the correlation between high expression of cIAP1 and poor
   prognosis in OSCC, we tested the efficacy of SMs in OSCC. OSCC cell
   lines were treated with monovalent SMs LCL161 or AT406. As observed in
   other tumor types [[133]41], OSCC cell lines exhibited differential
   sensitivity to SM treatment (Fig. [134]3A, B, Supplementary Fig.
   [135]4A, B). LCL161 was selected for further experiments due to its
   slightly superior efficacy compared to AT406 (Fig. [136]3A, B,
   Supplementary Fig. [137]4A, B). OSCC cell lines HSC-3 and HN6 were
   sensitive (Fig. [138]3A) and CAL-33, HSC-2, SCC-172, SCC-131, SCC-9,
   SCC-4 and CAL-27 were resistant (Fig. [139]3B) to LCL161 killing.
   Additionally, we found LCL161 was unable to induce human epidermal
   keratinocyte cell line (HACAT) death (Supplementary Fig. [140]4C).
   SM-sensitive HSC-3 and HN6, and SM-resistant CAL-33, CAL-27, and SCC-9
   cell lines were chosen as representatives for the majority of the
   following experiments in this study. Moreover, flow cytometry analysis
   using Annexin-V FITC/PI demonstrated that a higher proportion of HSC-3
   (Fig. [141]3C, E) and HN6 (Supplementary Fig. [142]4D, E) cells
   underwent early (AV^+/PI^−) and late apoptosis (AV^+/PI^+) compared to
   CAL-33 (Fig. [143]3D, F) following 24 h of LCL161 treatment. Mirroring
   the survival assays, crystal violet clonogenic survival assays showed
   that HSC-3 and HN6 cells were effectively killed by LCL161 treatment
   while few CAL-33 cells died (Fig. [144]3G, H, Supplementary Fig.
   [145]4F).

Fig. 3. OSCC tumor cells exhibit variable sensitivity to SM.

   [146]Fig. 3
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   Cell viability measured by CCK-8 of OSCC sensitive (A) or resistant (B)
   cell lines after treatment with SM LCL161 (0.5 or 1 μM) for 24 h, *
   P < 0.05, ** P < 0.01, *** P < 0.001, P = ANOVA analysis.
   Representative dot plots of the proportion of live (AV^−/PI^−), early
   apoptotic (AV^+/PI^−), and late apoptotic (AV^+/PI^+) in HSC-3 (C) and
   CAL-33 cells (D) post-treatment with SM LCL161 for 24 h. Bar graphs
   showing the percentage of HSC-3 (E) and CAL-33 cells (F) within each
   quadrant. Representative images showing crystal violet staining of
   HSC-3 (G) and CAL-33 (H) cells post-treatment for 24 h with/without 0.5
   or 1 μM LCL161. I Diagrammatic summary and timeline of the experimental
   protocol for the transplantation of OSCC cells into nude mice
   subcutaneously and treatment with vehicle or LCL161 (via
   intraperitoneal injection). Representative images of gross tumor
   lesions obtained from nude mice bearing with HSC-3 (J) or CAL-33 cell
   (K). Graphical representation of tumor volume (L, N) and mouse weight
   (M, O) from nude mice subcutaneously transplanted with HSC-3 or CAL-33
   cells, P = t-test. Error bars are Mean ± SD.

   To verify the efficiency of LCL161 in vivo, 4-week-old BALB/c nude
   female nude mice (GemPharmatech, China) were used to construct a
   subcutaneous OSCC xenograft model. 2 × 10^6 HSC-3 or CAL-33 cells were
   inoculated subcutaneously into the right armpit. (Fig. [148]3I). The
   progression of the HSC-3 subcutaneous xenograft model treated with
   LCL161 was significantly blunted in comparison to the vehicle group
   (Fig. [149]3J, L). In contrast, LCL161 treatment failed to reduce tumor
   growth in mice bearing CAL-33 xenografts, which is consistent with the
   in vitro data (Fig. [150]3K, N). These results indicated that OSCC
   tumor cell lines display differential sensitivities to SM treatment.
   Additionally, there was no difference in tumor recipient mouse body
   weight between the LCL161 treatment group and vehicle group (Fig.
   [151]3M, O). We subsequently performed hematoxylin and eosin (H&E)
   staining of the recipient mouse livers and spleens. We did not observe
   alterations in the pathologic features associated with drug injury,
   such as vacuolar degeneration of hepatocytes and enrichment of splenic
   lymphocytes in LCL161 treatment group. There was some indication of
   splenomegaly and disorganization of lymphoid follicles in the spleen
   after LCL161 treatment however this is consistent with the ability of
   SMs to activate non-canonical NF-κB (Supplementary Fig. [152]4G). These
   results demonstrate the dosing safety profile of LCL161 in vivo
   treatment.

cIAP1 is degraded upon SM treatment in both OSCC-sensitive and resistant
tumor cells

   Since OSCC cells showed differential responses to SM treatment, we
   sought to analyze the factors that might contribute to SM therapy
   resistance. First, the expression of cIAP1 and cIAP2 was assessed by
   western blot (WB). We found that both sensitive and resistant OSCC cell
   lines expressed cIAP1 pre SMs stimulation, but the expression levels
   did not obviously correlate with their sensitivity to SM therapy (Fig.
   [153]4A). Compared to cIAP1, cIAP2 expression was more variable, but
   again did not correlate with SM sensitivity (Fig. [154]4A). In summary,
   the ability of SM therapy to induce OSCC death did not appear to
   correlate with cIAP1 or cIAP2 expression levels.

Fig. 4. SM induces cIAP1 degradation in both SM-sensitive and resistant OSCC
tumor cells.

   [155]Fig. 4
   [156]Open in a new tab

   A Western blot showing original expression levels of cIAP1 and cIAP2
   for the indicated cell lines without any stimulation. Western blot
   showing expression levels of cIAP1 and cIAP2 pre and post-treatment
   with/without LCL161 1 μM (+) in SM-sensitive cell lines (B) and
   SM-resistant cell lines (C) for 24 h. Representative IHC images of
   cIAP1 (D) and cIAP2 (F) and graphical quantitation from tumor slides
   obtained from HSC-3 bearing mice, P = Student’s t-test. Representative
   IHC staining of cIAP1 (E) and cIAP2 (G) and graphical quantitation of
   tumor slides obtained from CAL-33 bearing mice, P = Student’s t-test.
   Error bars are Mean ± SD.

   Next, we assessed SM-induced degradation of cIAP1 and cIAP2 in OSCC
   cells. In this instance, cIAP1 levels were decreased after treatment
   with LCL161 for 24 h regardless of whether the cells were SM-sensitive
   or not (Fig. [157]4B, C). In comparison, cIAP2 expression remained low
   and unaltered (Fig. [158]4B, C). To determine whether SM treatment
   elicited a similar pharmacological response in vivo, we performed IHC
   staining for cIAP1 and cIAP2 in BALB/c nude mice subcutaneously grafted
   with HSC-3 or CAL-33 cells and treated with LCL161. Consistent with our
   in vitro data, both HSC-3 and CAL-33 transplanted mice showed reduced
   expression levels of cIAP1 (P = 0.0003, P = 0.0146) (Fig. [159]4D, E)
   but not cIAP2 after treatment with LCL161 (Fig. [160]4F, G). These
   results demonstrated that LCL161 successfully induced cIAP1 degradation
   in both the HSC-3 and CAL-33 cell xenograft models after treatment with
   LCL161.

Addition of exogenous TNF reverses SM resistance in a subset of OSCC cells

   SMs induce cell death as a single agent by simultaneously inducing
   autocrine TNF production and sensitizing cells to TNF killing [[161]13,
   [162]42]. We therefore measured the TNF concentrations in both
   SM-sensitive and resistant OSCC cells using ELISA. Surprisingly, all
   tested cells had detectable basal TNF expression (Fig. [163]5A) in the
   absence of any stimulation. However, the SM-sensitive cells secreted a
   comparatively higher concentration of TNF compared to that of
   SM-resistant cells, excepting CAL-27 cells (Fig. [164]5A). Since
   SMs-induced cell death is correlated with TNF concentration [[165]43,
   [166]44], we also tested the TNF concentration after LCL161 treatment
   via ELISA. Interestingly, we only observed a significant upregulation
   of autocrine TNF in SM-sensitive HSC-3 and HN6 cells after 24 h,
   although approximately 50% of the cells were apoptotic at this time
   (Supplementary Fig. [167]5A, Fig. [168]3A).

Fig. 5. OSCC-resistant cells fail to form Complex IIa.

   [169]Fig. 5
   [170]Open in a new tab

   A Graphical representation of Elisa autocrine TNF measurements from
   nine OSCC cell lines. * P < 0.05, ** P < 0.01, *** P < 0.001, ***
   P < 0.0001, P = ANOVA analysis. Compared with the TNF concentration of
   HSC-3: mark ‘*’ in black, compared with the TNF concentration of HN6:
   mark ‘*’ in red. B Graphical representation of the percentage viability
   of OSCC cell lines following treatment with LCL161 combined with TNF
   (10 ng/mL), * P < 0.05, ** P < 0.01, *** P < 0.001, P = ANOVA analysis.
   Assessment of Complex IIa formation by immunoprecipitation for RIP1,
   FADD, and caspase-8 in HSC-3 (C) and CAL-33 (D) after LCL161 treatment.
   Western blot detection for the cleavage of caspase-8 (E) and caspase-3
   (F) in HSC-3 and CAL-33 after LCL161 stimulation. Representative images
   and quantitative analysis of IHC staining for cleaved-caspase-3 from
   tumors isolated from HSC-3 (G) or CAL-33 (H) bearing nude mice.
   P = Student’s t-test. Error bars are Mean ± SD.

   Previous studies have reported that resistance to SM therapy can be
   overcome by upregulation of TNF either directly or indirectly [[171]24,
   [172]45, [173]46]. To exclude the possibility that insufficient TNF
   leads to SM resistance, we treated all OSCC cells with or without high
   concentrations of exogenous TNF plus LCL161. However, while the
   addition of TNF had a minor effect in reversing LCL161 resistance in
   SCC-9, SCC-4, and CAL-27 cells, it had no effect on the four other OSCC
   cell lines (Fig. [174]5B). Therefore, while TNF was important for
   promoting SM-mediated cell death, it did not appear to be the key
   factor conferring resistance to SM treatment in OSCC cells.

OSCC-resistant cells fail to form Complex IIa after SM treatment

   Since we found that both resistant and sensitive cells produce TNF, we
   examined whether they assembled Complex IIa in response to SM
   treatment. Consistent with the death data, LCL161 induced a
   RIPK1-caspase-8 containing complex in SM-sensitive HSC-3 but not CAL-33
   cells (Fig. [175]5C, D). Cleavage of caspase-8 and cleavage of
   caspase-3 are indicators of SM-induced activation of apoptotic pathway
   [[176]13, [177]29], and consistently, SM treatment promoted the
   increased cleavage of total caspase-8 and caspase-3 protein in
   SM-sensitive HSC-3 cells compared to SM-resistant CAL-33 cells (Fig.
   [178]5E, F). We also sought to verify this finding in vivo by staining
   for cleaved-caspase-3 in our subcutaneous xenograft models. Consistent
   with our previous results, cleavage of caspase-3 was only observed in
   SM-sensitive HSC-3 subcutaneous xenograft mice treated with LCL161
   (Fig. [179]5G) but not in mice bearing SM-resistant CAL-33 (Fig.
   [180]5H).

SMs activate the non-canonical NF-κB pathway in both SM-sensitive and
resistant OSCC cells

   In addition to modulating cell death, SMs affect cell survival via
   opposing effects on the canonical (NF-κB1/RelA) and non-canonical
   (NF-κB2/RelB) NF-κB pathways [[181]13]. Specifically, SMs prevent
   TNF-induced activation of the canonical NF-κB pathway and divert the
   pro-survival complex to a death complex. On the other hand, SMs
   activate the non-canonical NF-κB pathway by inhibiting cIAP-induced
   degradation of the non-canonical NF-κB activating kinase (NIK)
   [[182]47–[183]49]. To determine whether activation of non-canonical
   NF-κB might contribute to resistance, we examined the activation of
   non-canonical NF-κB in 5 OSCC cell lines after LCL161 treatment for
   24 h. Consistent with previous reports [[184]50], nuclear-localized p52
   (NF-κB2) levels were significantly increased (Fig. [185]6A). Consistent
   with the in vitro studies, both SM-sensitive HSC-3 and SM-resistant
   CAL-33 xenografts showed higher IHC score of NIK after LCL161 injection
   (Fig. [186]6B, C). We then investigated NF-κB1 activation, although
   cytoplasmic localized unprocessed p105 expression levels, which were
   downregulated, with no significant change in nuclear p50 (NF-κB1)
   observed in each of the 5 OSCC cell lines after treatment with LCL161
   for 24 h (Fig. [187]6D).

Fig. 6. SM activates non-canonical NF-κB pathway in both SM-sensitive and
resistant OSCC cells.

   [188]Fig. 6
   [189]Open in a new tab

   A Representative WB of total, cytoplasmic, and nuclear fractions of
   NF-κB2 (p100 and p52) in HSC-3, HN6, CAL-33, CAL-27, and SCC-9 OSCC
   cell lines treated with LCL161 for 24 hours. Representative images of
   IHC staining for NIK protein and quantitative analysis from tumor
   tissue isolated from HSC-3 (B) or CAL-33 (C) bearing nude mice treated
   with Vehicle or LCL161, P = Student’s t-test. Error bars are Mean ± SD.
   D Representative western blots of total, cytoplasmic, and nuclear
   fractions of NF-κB1 (p105 and p50) in HSC-3, HN6, CAL-33, CAL-27, and
   SCC-9 OSCC cell lines after 24 h treatment with LCL161.

ABCA3, ABCB1, and ABCB4 upregulation in OSCC cells contributes to SM
resistance

   An increasing number of studies have suggested that the IAP family
   regulates cellular metabolism [[190]51, [191]52]. After excluding
   well-known factors (cIAPs levels, TNF concentration, and NF-κB pathway
   activation) as determinants of the differential sensitivity of OSCC
   cells to SM treatment, we performed gas chromatography-mass
   spectrometry (GC-MS) to identify metabolic differences between
   SM-sensitive and resistant cells after SM stimulation for 6 h. A total
   of 27 metabolites were found to be significantly differentially
   expressed between sensitive HSC-3 and HSC-3 plus LCL161 (Fig. [192]7A),
   while 28 metabolites were found to be significantly different between
   resistant CAL-33 and CAL-33 plus LCL161 (Fig. [193]7B). Only 3
   metabolites overlapped: tagatose, aspartic acid, and uridine 3TMS
   major, indicating that the HSC-3 treated group didn’t share a similar
   metabolic trend with the CAL-33 treated group (Fig. [194]7C). Moreover,
   the basal metabolites of HSC-3 and CAL-33 were undoubtedly different
   (Supplementary Fig. [195]6A).

Fig. 7. SM-resistant cells exhibited enrichment of the ABC transporter
pathway.

   [196]Fig. 7
   [197]Open in a new tab

   Heatmaps showing differential metabolite expression (variable
   importance for projection >1, P < 0.05) in HSC-3 (A) and CAL-33 (B)
   OSCC cell lines post control (Ctrl) or treatment with 1 μM LCL161 for
   6 h. C Venn diagram showing the numbers of overlapping and
   differentially expressed metabolites in HSC-3 and CAL-33 OSCC cell
   lines post LCL161 treatment. KEGG pathway enrichment analysis of
   differential metabolites for HSC-3 (D) and CAL-33 (E) cells post LCL161
   treatment for 6 h.

   A KEGG pathway enrichment analysis found that LCL161 treatment was
   associated with a signature for unsaturated fatty acids biosynthesis in
   HSC-3 (Fig. [198]7D), whereas this treatment resulted in a signature
   for ABC transporter pathway activation in CAL-33 (Fig. [199]7E).
   Although the mRNA levels of several ABC transporter family members were
   not significantly increased in the SM-resistant CAL-33, CAL-27, and
   SCC-9 cell lines, after LCL161 treatment (Supplementary Fig.
   [200]7A-E), mRNA levels of ABCA3, ABCB1, and ABCB4 were higher in the
   SM-resistant cells than in the sensitive HSC-3 in the absence of any
   stimulation (Fig. [201]8A). The upregulation of ABCA3 in CAL-33
   subcutaneous xenografts (P = 0.0546) was more pronounced than in HSC-3
   subcutaneous xenografts (P = 0.1094) after LCL161 injection (Fig.
   [202]8B, C), but it was not statistically significant, which was
   consistent with the results of mRNA expression analysis (Supplementary
   Fig. [203]7A, C).

Fig. 8. Upregulation of the ABC transporter proteins ABCA3, ABCB1, and ABCB4
in OSCC cells contributes to SM resistance.

   [204]Fig. 8
   [205]Open in a new tab

   A Heatmap of showing basal transcription levels of the ABC transporter
   families from CAL-33, CAL-27, and SCC-9 OSCC cells, with HSC-3 used as
   control. Representative images of IHC staining for ABCA3 protein and
   quantitative analysis from tumor tissue isolated from HSC-3 (B) or
   CAL-33 (C) subcutaneous xenograft tumor model treated with Vehicle or
   LCL161, P = Student’s t-test. Graphical representation of the viability
   percentage of CAL-33 (D) and SCC-9 (E) OSCC cell lines following the
   indicated treatments. * P < 0.05, ** P < 0.01, *** P < 0.001. P = ANOVA
   analysis. Compared with the control group: mark ‘*’ in black, compared
   with the group treated with 1 μM LCL161: mark ‘*’ in red, compared with
   the group treated with 1 μM LCL161 and 10 ng/mL TNF: mark ‘*’ in blue,
   compared with the group treated with verapamil: mark ‘*’ in pink,
   compared with the group treated with genistein: mark ‘*’ in yellow.
   mRNA expression of ABCA3, ABCB1 and ABCB4 from SM-sensitive OSCC cell
   lines CAL-33 (F), CAL-27 (G) and SCC-9 (H) after knockdown with siRNA.
   Protein expression of ABCA3 (I), ABCB1 (J), and ABCB4 (K) from
   SM-sensitive OSCC cell lines CAL-33, CAL-27, and SCC-9 after knockdown
   with siRNA. Graphical representation of the viability percentage of
   CAL-33 (L), CAL-27 (M), SCC-9 (N) OSCC cell lines with
   siABCA3/siABCB1/siABCB4 following the indicated treatments. Compared
   with the relative controls: * P < 0.05, ** P < 0.01, *** P < 0.001.
   P = ANOVA analysis. O Representative western blots of cIAP1 expression
   of CAL-33, CAL-27, and SCC-9 treated with LCL161 combined with
   genistein (5 μM) or verapamil (5 μM) for 0-2 h. P Representative
   western blots of cIAP1 expression of HSC-3, CAL-33, and CAL-27 treated
   with LCL161 for 0–1 h. Error bars are Mean ± SD.

   Based on these findings, we speculated that SM-resistant OSCC cells
   might efflux SMs to gain resistance. Therefore, in addition to SM
   treatment, we also inhibited ABC transporters using the ABCA3
   inhibitor, genistein [[206]53], and the ABCB1 and ABCB4 inhibitor,
   verapamil [[207]54–[208]56] and evaluated their ability to
   synergistically induce cell death in OSCC cell lines. Consistent with
   the idea that ABC transporters are more active in resistant cells,
   LCL161 in combination with ABC inhibitors induced more cell death than
   LCL161 alone in SM-resistant CAL-33(Fig. [209]8D). For SCC-9, the
   addition of exogenous TNF makes this combination regimen effective
   (Fig. [210]8E). To further validate the hypothesis, we subsequently
   targeted knockdown of ABCA3, ABCB1and ABCB4 using siRNA (Fig.
   [211]8F-K) and found that SM-resistant cell lines after silencing
   ABCA3, ABCB1 and ABCB4 were more sensitive to SM-induced death (Fig.
   [212]8L-N). Supporting the idea that LCL161 is an ABC transporter
   substrate that is actively transported out of the cell. Consistently,
   we also observed that LCL161, in combination with ABC transporter
   inhibitors, induced more complete degradation of cIAP1 in SM-resistant
   cell lines (Fig. [213]8O), but did not influence SM-sensitive cell
   lines (Supplementary Fig. [214]8A). In addition, we compared the extent
   of cIAP1 degradation in SM-sensitive and resistant cells and found that
   although there was no significant difference in cIAP1 degradation in
   the two sub-type cell lines after 24 h of LCL161 stimulation (Fig.
   [215]4B, C), resistant cells treated with LCL161 for a short time
   (0–1 h) showed inexhaustive degradation of cIAP1 and even a gradual
   increase in the expression of cIAP1, while the sensitive cells
   displayed decreased expression of cIAP1 (Fig. [216]8P). The degradation
   of cIAP1 by SMs, particularly during the first few hours of treatment,
   appeared to correlate with OSCC sensitivity to SMs.

Discussion

   While SMs have been proven to be safe, even in combination with other
   cytotoxic drugs, they have not until recently, dramatically improved
   treatment outcomes [[217]13]. In recent trials where the SM xevinapant
   was combined with standard-of-care chemo-radiotherapy in HNSCC,
   dramatic improvements were observed: the risk of death or disease
   progression was reduced by 67% [[218]32, [219]33]. These results
   provide a strong impetus to discover why HNSCCs, of which OSCCs are an
   important subset, respond to SM treatment.

   We considered that IAP expression levels might correlate with
   sensitivity to SMs [[220]57, [221]58], and therefore assessed IAP
   expression levels in OSCC. Approximately 5–9% of OSCCs contain
   amplifications of BIRC2 (encoding cIAP1) and BIRC3 (encoding cIAP2)
   locus 11q22.1-11q22.2, both in the TCGA database and from 88 local
   patients at the DNA level. Note that the BIRC2 and BIRC3 copy number we
   measured here are not Copy Number Variations (CNVs). They only predict
   an increased number of copies, and there may be false positives and
   false negatives. The expression level of cIAP2 protein was also higher
   in tumors. However, comparison of cIAP2 mRNA levels between tumor and
   normal counterparts were not significantly different in the TCGA
   cohort. This discrepancy may be due to different patient cohorts being
   assessed. In the present study, we were unable to obtain mRNA from the
   OSCC patient cohort, therefore we used an IHC approach to measure
   protein expression levels. Further studies assessing cIAP2 mRNA and
   protein levels in OSCC are required to confirm if this discrepancy was
   due to the TCGA cohort containing HNSCC samples rather than wholly
   OSCC. High transcription level of BIRC2 was predictive of poorer
   patient outcomes, as was higher cIAP1 protein expression in tumors,
   compared to paired non-tumor counterparts. However, high cIAP2 was
   actually predictive of a better outcome. cIAP2 expression is increased
   upon SM treatment most likely as a result of non-canonical NF-κB
   activation caused by cIAP1 degradation [[222]59]. Therefore, it is
   plausible that high levels of cIAP2 indicate low levels of cIAP1 and
   that these two observations are consistent with each other. While the
   SMs we used in our study target cIAP1(BIRC2), cIAP2 (BIRC3) and XIAP
   (BIRC4), we did not examine XIAP levels in our resistant and sensitive
   cell lines and it, therefore, remains possible that XIAP levels could
   affect sensitivity. Nevertheless, mutations affecting XIAP or caspase-9
   occur rarely in HNSCC compared to cIAP1/2 [[223]60, [224]61] and
   extrinsic apoptosis effectors such as FADD and caspase-8, suggesting
   that XIAP and the intrinsic pathway regulated by it are not as
   important in HNSCC.

   TNF is required for SM mediated cell death [[225]14, [226]15], and
   overall we observed a correlation between sensitivity to SM and TNF
   expression, even though one of the seven resistant cell lines expressed
   TNF to similar levels as the two sensitive cell lines. While the
   transcription factor specificity protein 3 (SP3) has been shown to
   confer differential sensitivity to SM by regulating TNF expression
   [[227]62]. However, TNF production was not the only limiting factor,
   since addition of exogenous TNF only partially reversed resistance to
   SM treatment in three of the resistant cell lines. In addition, all
   OSCC cell lines tested secreted TNF, and while the two sensitive lines
   secreted the highest levels of TNF, one of the resistant lines secreted
   very similar amounts as these two. However, only sensitive cells
   increased secretion of TNF upon SM treatment by twofold or more.
   Notably, non-canonical NF-κB can contribute to TNF production and this
   pathway was also activated in both SM-sensitive and resistant cells.
   The SIX transcriptional repressors have also been shown to suppress SM
   mediated death [[228]63], by inhibiting non-canonical NF-κB activation
   through NF-κB responsive promoters. Therefore, it is possible that
   differences in SIX expressions could influence and inhibit SM-induced
   death post p100 processing and p52 translocation into the nucleus.

   The ABC transporter family is responsible for multi-drug resistance
   through drug efflux [[229]64]. Clinical ABCB1 inhibitors enhance the
   bioavailability of a number of different SMs [[230]65], and we have
   shown that LCL161 is a direct substrate of ABCB1 [[231]66]. In this
   study, we found that SM-sensitive cells had uniformly low levels of ABC
   family members while resistant cells had high levels of ABCA3, ABCB1,
   and ABCB4. These seem to contribute to LCL161 resistance since
   co-treatment with verapamil (an ABCB1 and ABCB4 inhibitor) or genistein
   (an ABCA3 inhibitor) protected them from apoptosis. Currently, ABC
   transporter inhibitors have not been successfully deployed in the
   clinic to overcome multidrug resistance. However, they have usually
   been combined with cytotoxic drugs, rather than drugs like SMs, which
   are considerably less toxic to healthy cells [[232]67]. Indeed the SM,
   ABC transporter inhibitor combination was well tolerated and
   efficacious in treating animal models of infection and leukemia
   [[233]44, [234]65]. Furthermore, the ABC transporter inhibitor,
   lazertinib was found to improve the efficacy of chemotherapy in
   patients with ABCB1 or ABCG2 overexpression [[235]68]. Therefore, the
   combination of SMs and lazertinib could have promising therapeutic
   potential for OSCC patients with high ABC transporter expression.

   In summary, our study fills a gap in knowledge relating to how SM drug
   resistance arises and potentially how it can be overcome to improve
   outcomes for OSCC. We assessed a considerable number of clinical OSCC
   samples to analyze the correlation between cIAP expression and OSCC
   patients’ prognosis. Our clinical sample data indicates that patients
   with OSCC that overexpress cIAP1 have lower survival rates, suggesting
   cIAP1 is a valid therapeutic target, especially for SMs that promote
   cIAP1 destruction. However, degradation of cIAP1 is associated with a
   number of outcomes, including non-canonical NF-κB activation and, in
   some cells, TNF production which can either combine to kill cells or in
   some cases make them more resistant to cell death. Thus, for example,
   in lymphomas, activation of non-canonical NF-κB is a strong survival
   signal, and therefore mice with Myc lymphomas treated with LCL161 have
   decreased survival [[236]69]. Furthermore, while for some cells SM
   induced autocrine production of TNF and loss of cIAP1 is sufficient to
   induce apoptosis [[237]14, [238]15], our data show that for a subset of
   OSCC, even the addition of exogenous TNF is not sufficient to induce
   death in resistant cells. This is despite the fact that these resistant
   OSCC cells nevertheless express many of the extrinsic apoptosis
   effector molecules, including RIP1 FADD, caspase-8, and -3. However,
   the functional expression of these proteins was undetermined and more
   proteins have been reported to regulate formation or activity of the
   death-inducting signaling complex. Therefore, the basis of their
   resistance is still unclear. Finally, some, but not all SMs, are
   substrates of ABC transporters providing another route to resistance.
   This is particularly relevant to cancer cells that have already been
   subjected to cytotoxic therapy as resistance to some cytotoxic
   therapies can be driven by upregulation of ABC transporters [[239]70].
   Some of these resistance mechanisms can be accommodated, particularly
   if they’re known in advance. Nevertheless, in all likelihood SMs will,
   as in the recent HNSCC trials, be combined with chemo and or
   radiotherapy which may provide some of the necessary signaling to
   overcome resistance by, for example, upregulating TNF production and
   driving extrinsic apoptotic signaling independently of TNF [[240]71].
   Thus our findings may help improve outcomes for OSCC patients and other
   sub-groups of HNSCC [[241]34] and potentially other solid cancers with
   elevated cIAPs, such as cervical, ovarian and bladder [[242]19].

Materials and methods

Human ethics and sample information

   All research complied with the relevant ethical regulations. OSCC
   patient samples and information were acquired with patient informed
   consent and handled within the guidelines and policies of the Research
   Ethics Committee of Nanjing Stomatology Hospital (NJSH-2021NL-026).

   A total of 62 patients were included for cIAP1 IHC staining, and 71
   patients were included for cIAP2 IHC staining. Tumor tissues and
   corresponding normal tissues were collected from 187 patients for DNA
   amplification mutation analysis. 88 of these samples were used for
   transcriptional levels analysis of BIRC2 and BIRC3.

   The inclusion criteria were: ① OSCC diagnosed by postoperative
   pathology; ② primary case; ③ no other treatment before surgery; ④
   complete information on pathology and clinical follow-up. The exclusion
   criteria were: ① history of other malignant tumors; ② death due to
   reasons unrelated to OSCC. All samples and information were obtained
   from Affiliated Stomatological Hospital, Medical School of Nanjing
   University.

Mice

   4-week-old female BALB/c nude mice were purchased from GemPharmatech,
   Jiangsu, China. All mice were housed as follows: 12 light/12 dark
   cycles; temperature 28 °C, humidity 40–50%. All animal experiments were
   performed in accordance with the Jiangsu Association for Laboratory
   Animal Science protocols and subjected to review by the animal welfare
   and ethical review board of Nanjing University (IACUC-D2103002).

Reagents and antibodies

   Smac mimetics (LCL161, AT406) and ABC transporter inhibitors
   (genistein, verapamil) were purchased from Selleck, Shanghai, China.
   Additional information relating to the antibodies is listed in
   Supplementary Table [243]5.

Cell culture

   The cell lines used in this study were purchased from ATCC. All the
   cells were professionally verified to exclude mycoplasma or other
   contaminants. All cells were cultured in high glucose, Dulbecco’s
   Modified Eagle Medium (Thermo Fisher Scientific Inc, Massachusetts,
   USA) supplemented with 10% fetal bovine serum (Bio-Channel, Jiangsu,
   China) and 1% penicillin-streptomycin (New Cell and Molecular Biotech,
   Jiangsu, China). Cells were incubated at 37 °C in a standard humidified
   atmosphere of 5% CO2.

DNA mutation analysis in TCGA

   The cBIOPortal website ([244]http://www.cbioportal.org/) for cancer
   genomics was used for the analysis of BIRC2 and BIRC3 gene mutations.
   These levels are derived from copy-number analysis algorithms like
   GISTIC or RAE. Copy number analysis indicates copy-number level per
   gene, in which “−2” is a deep loss (homozygous deletion), “−1” is a
   shallow loss (heterozygous deletion), “0” is diploid, “1” indicates a
   low-level gain, and “2” is a high-level amplification [[245]34].

DNA copy number analysis by qPCR

   Copy number analyses of BIRC2 and BIRC3 were quantified by qPCR using
   the ViiA™ 7 Real-Time PCR System (Thermo Fisher Scientific Inc,
   Massachusetts, USA). Globin was used as the single-copy reference
   control and samples were assessed in triplicate. Primer sequences are
   listed in Supplementary Table [246]2. The SYBR Green Premix Pro Taq HS
   qPCR Kit (Accurate Biology, Hunan, China) was used according to the
   manufacturer’s instructions. PCR conditions: Step 1, 95 °C for 30 s for
   one cycle; Step 2, 95 °C for 5 s, 55 °C for 30 s, 72 °C for 30 s,
   repeated for 40 cycles; Step 3, dissociation. Copy number analysis of
   BIRC2 and BIRC3 was carried out using the comparative Ct method after
   validating that the efficiencies of PCR reactions of reference controls
   and genes under investigation are equal. Greater than 4 times gene copy
   number compared to reference control was deemed indicative of gene
   amplification.

Mouse subcutaneous xenograft OSCC model

   2 × 10^6 HSC-3 or CAL-33 cells were inoculated subcutaneously into the
   right armpit. 10 days post-inoculation, when the tumor volume was about
   150 mm^3 (Tumor volume = length × width × width/2), mice were divided
   into Vehicle group or SM (LCL161) treatment group randomly. Each group
   had a minimum of at least 10 mice and average initial tumor size
   between groups was similar. LCL161 was prepared by dissolving in 0.1 N
   hydrochloric acid, followed by dilution in 0.1 N anhydrous sodium
   acetate (Solarbio, Beijing, China) to 70% of the total volume. Vehicle
   (30% 0.1 N hydrochloric acid + 70% 0.1 N anhydrous sodium acetate
   solution) or LCL161 (20 mg/kg) were injected intraperitoneally every
   two days, with five repeats. Body weight and tumor volumes were
   recorded during the experiment. Once the tumor volume exceeded
   1500 mm^3, or the tumor surface skin broke down, or the mice lost more
   than 20% of body weight, the mice were withdrawn from the experiment
   and were euthanized immediately. On the 20th day post OSCC inoculation,
   mice were euthanized and tumor tissues were collected. During the
   process of this experiment, no blinding was done.

Cell viability assay: CCK-8

   OSCC cell lines were dispersed into 96-well plates at 5000 cells per
   well, cultured overnight, followed by treatment with 0, 0.5 μM or 1 μM
   LCL161, or 8 μM AT406 combined with/without 10 ng/mL TNF (Peprotech,
   Massachusetts, USA) for 24 hours. Media was then supplemented with a
   10:1 ratio of CCK-8 solution (Vazyme, Jiangsu, China) followed by
   culture for an additional 1–2 h. Absorbance values were read on a
   microplate reader (Molecular Devices, California, USA) at 450 nm
   wavelength. Plate reads were recorded when the control group absorbance
   value reached 0.8–1.2. Cell viability was calculated using the formula
   [(A-C)/(B-C)] × 100%, where A = experimental, B = control, C = blank
   (absorbance value of media and CCK-8 Solution only, without cells).

Crystal violet staining

   Following CCK-8 analysis, media was discarded and cells were fixed with
   4% paraformaldehyde for 30 min, followed by washing with PBS
   (3 × 5 min). Crystal violet (50 μL 0.5%) was then added to each well,
   incubated for 15 min, followed by washing with PBS (3 × 5 min). Plates
   were dried at 37 °C and data recorded by microscopy photos.

Flow cytometry

   HSC-3 or CAL-33 cells were dispersed into 12-well plates and allowed to
   adhere overnight, followed by treatment with 0, 0.5 μM, or 1 μM LCL161
   combined with/ without 10 ng/mL TNF for 24 h. Cell supernatants were
   collected and combined with single cell suspensions of trypsinsed cells
   (EDTA-free trypsin) and stained using Annexin-V FITC and PI apoptosis
   detection kit (Vazyme, Jiangsu, China). The data were acquired by FACs
   Calibur (BD Biosciences, New Jersey, USA) and analyzed using Treestar
   FlowJo software.

Western blot

   Treated OSCC cells were lysed at 4 °C for 30 min in RIPA buffer
   (Beyotime, China), containing phosphatase (Phosstop^TM, Roche, Basel,
   Switzerland) and protease inhibitors (Complete^TM, Roche, Basel,
   Switzerland). Samples were then centrifuged and the supernatant mixed
   with SDS-PAGE loading buffer, heated at 95 °C for 10 min and the
   extracted proteins were analysed by western blot using 4–20% SDS-PAGE
   (Smart Life-science, Jiangsu, China), followed by transferring to PVDF
   membrane (Millipore, Massachusetts, USA). Blots were blocked with 3%
   BSA dissolved in 1x TBST. Primary antibodies were incubated overnight
   on a shaker at 4 °C, followed by incubation with HRP-conjugated
   secondary antibodies for 45 min at room temperature and visualized by
   Enhanced Chemiluminescence (Vazyme, Jiangsu, China). Cytoplasmic and
   nuclear extracts from OSCC cell lines were prepared using NE-PER
   extraction reagents (Thermo Fisher Scientific Inc, Massachusetts, USA)
   according to the manufacturer’s instructions. Original data of WB are
   reported as Original Data 1.

Immunoprecipitation (IP)

   Protein A/G beads were washed 3 times with PBS containing 0.5% Triton,
   separated magnetically with a magnetic rack, and resuspended in 400 µL
   of PBS containing 0.5% Triton. Washed A/G beads were mixed with
   antibodies for IP for 1 h at room temperature, washed with PBS
   containing 0.5% Triton for 3 times, and then incubated with cell
   lysates for an additional 1 h at room temperature, followed by
   separation using DynaMag™-2 magnets (Thermo Fisher Scientific Inc,
   Massachusetts, USA). Beads were then washed 5 times with PBS containing
   0.5% Triton and eluted with 20 μL of 1x SDS sample buffer.

Histological preparation and IHC

   Tumor tissues were fixed in 4% paraformaldehyde for a minimum of 2 days
   before processing for paraffin embedding. Tissue blocks were sectioned
   (5 μm), deparaffinized, rehydrated, and subjected to heat-induced
   epitope retrieval with citrate buffer, pH 6.0 (Zhongshan Golden Bridge,
   China), and blocked with goat serum (Zhongshan Golden Bridge, Beijing,
   China). Sections were stained with primary antibodies at 4 °C
   overnight, washed with 1x PBST for 5 min (×3), followed by incubation
   with anti-Mouse/Rabbit generic secondary antibodies (Maxin, Fujian,
   China) for 30 min at room temperature. Slides were then washed with 1x
   PBST for 5 min (×3) followed by incubation with DAB kit (Zhongshan
   Golden Bridge, Beijing, Chian) according to the manufacturer’s
   instructions. Then sections were counterstained, differentiated, and
   reblued in hematoxylin, 10% ethanol hydrochloride, and alkaline reblue
   solution, respectively. At last, sections were dehydrated and sealed.

   Protein expression was evaluated according to a stain intensity score
   (0 = negative; 1 = weak; 2 = moderate; 3 = high) and a positive
   percentage score (0 = no positive staining, 1 = 1–25%, 2 = 26–50%,
   3 = 51–75%, 4 = 76–100% positive cells). Two independent experienced
   pathologists performed the IHC scoring. Defined by the median final
   score, the expression levels of proteins were defined as “low” or
   “high”. Pathologists were blind to follow-up information about
   patients.

H&E

   Similar to the protocol of IHC, the liver and spleen tissues of mice
   were fixed to prepare paraffin blocks. Tissue blocks were sectioned (5
   μm), deparaffinized, and rehydrated. Then sections were counterstained,
   differentiated, and reblued as IHC experiment, followed by staining
   with eosin solution. Finally, sections were dehydrated and then sealed.

Quantitative real-time PCR

   RNA from OSCC cell lines: HSC-3, HN6, CAL-33, CAL-27, and SCC-9 treated
   with/without 1 μM LCL161 for 24 h was extracted using the SteadyPure
   RNA Extraction Kit (Accurate Biology, Hunan, China) according to the
   manufacturer’s instructions. Detection of mRNA levels of ABC
   transporters followed the protocol for DNA copy number, including PCR
   conditions of triplicate samples. GAPDH was chosen as the reference
   RNA. The qPCR data was analyzed using the 2-delta delta CT method.
   Primer sequences were listed in Supplementary Table [247]6.

ELISA

   2 × 10^5 OSCC cells were seeded into 60 mm dishes and cultured with the
   indicated treaments for 24 h. Supernatants were then centrifuged at
   1200 r/min for 5 min to collect supernatants. A human TNF-alpha ELISA
   Kit (Proteintech, Wuhan, China) was used to detect TNF concentration
   according to the manufacturer’s instructions. Absorbance at 450 nm was
   measured using microplate reader (Molecular Devices, California, USA)
   and a four-parameter logistic curve fit was used to calculate TNF
   concentration.

GC-MS analysis

   HSC-3 and CAL-33 were treated with LCL161 for 6 h, followed by snap
   freezing with liquid nitrogen. Once liquid nitrogen had evaporated,
   500 μL ddH[2]O was added to the culture plate. Samples were stored at
   −80 °C until GC-MS was performed. Data analysis was performed by
   Shengzi Biotech using methodology previously described [[248]72].

siRNA knockdown of ABCA3, ABCB1 and ABCB4

   To knockdown ABCA3, ABCB1, and ABCB4 in SM-resistant cell lines,
   siRNA-ABCA3, siRNA-ABCB1, and siRNA-ABCB4 were designed and transfected
   into CAL-33, CAL-27, and SCC-9 with the lipofectamine 3000 transfection
   kit (ThermoFisher, Massachusetts, USA). The siRNA sequences had been
   listed in Supplementary Table [249]7.

Statistical analysis

   Data were presented as mean ± SD. P values were calculated by Student’s
   t-test, paired t-test, ANOVA test, chi-square test, or log-rank
   (Mantel–Cox) test, the P values were two-tailed test if no otherwise
   specified. Normal distribution test and homogeneity of variance test
   were performed if needed. Statistical analysis methods were listed in
   the figure captions. All results have been validated by three or more
   independent experiments.

Supplementary information

   [250]Supplementary Figures and Tables^ (3.4MB, pdf)
   [251]Original Data 1^ (2.9MB, pdf)

Author contributions

   QH, YN, and XZ conceived the work; YW, ZJL, QS, WL, XZ, WJ, XY, and XH
   acquired data; YW, XZ, and LD interpreted data; PW, YS, and YJ managed
   biobank and follow-up information; YW, XZ, QY, and YN drafted the
   manuscript; XZ, YW, QS, ZJL, and ZHL revised the manuscript; LOR and JS
   provided intellectual input, helped interpret data and co-wrote the
   manuscript.

Funding

   This research was funded by the National Natural Science Foundation of
   China (No. 82173159, 82002865, 82173380), Key Research and Development
   Projects in Jiangsu Province (No. BE2020628), “3456” Cultivation
   Program for Junior Talents of Nanjing Stomatological Hospital, the
   Medical School of Nanjing University (No. 0222R216), the Nanjing
   Medical Science and Technology Development Foundation, Nanjing
   Department of Health (No. YKK20151, [252]QRX17083), Nanjing Health
   Science and Technology Development Major Project (No. ZDX22001) and
   NHMRC Investigator Grant (1195038).

Data availability

   The data supporting the findings of this study are available from the
   corresponding author upon reasonable request.

Competing interests

   The authors declare no competing interests.

Footnotes

   Edited by Kristopher Sarosiek

   Publisher’s note Springer Nature remains neutral with regard to
   jurisdictional claims in published maps and institutional affiliations.

Contributor Information

   Xiaoxin Zhang, Email: 693216371@qq.com.

   Qingang Hu, Email: qghu@nju.edu.cn.

   Yanhong Ni, Email: Yanhong.Ni@nju.edu.cn.

Supplementary information

   The online version contains supplementary material available at
   10.1038/s41419-024-07253-w.

References