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Pharmacological disruption of the MTDH–SND1 complex enhances tumor antigen presentation and synergizes with anti-PD-1 therapy in metastatic breast cancer

Nature Cancer volume 3, pages 60–74 (2022)Cite this article

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Abstract

Despite increased overall survival rates, curative options for metastatic breast cancer remain limited. We have previously shown that metadherin (MTDH) is frequently overexpressed in poor prognosis breast cancer, where it promotes metastasis and therapy resistance through its interaction with staphylococcal nuclease domain-containing 1 (SND1). Through genetic and pharmacological targeting of the MTDH–SND1 interaction, we reveal a key role for this complex in suppressing antitumor T cell responses in breast cancer. The MTDH–SND1 complex reduces tumor antigen presentation and inhibits T cell infiltration and activation by binding to and destabilizing Tap1/2 messenger RNAs, which encode key components of the antigen-presentation machinery. Following small-molecule compound C26-A6 treatment to disrupt the MTDH–SND1 complex, we showed enhanced immune surveillance and sensitivity to anti-programmed cell death protein 1 therapy in preclinical models of metastatic breast cancer, in support of this combination therapy as a viable approach to increase immune-checkpoint blockade therapy responses in metastatic breast cancer.

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Fig. 1: MTDH promotes metastatic breast cancer by enhancing immune evasion.
Fig. 2: MTDH inhibits T cell activation.
Fig. 3: MTDH destabilizes Tap1/2.
Fig. 4: MTDH forms a complex with SND1 to inhibit tumor antigen presentation and T cell activation.
Fig. 5: MTDH–SND1 blocking activates T cells by enhancing antigen presentation in tumors.
Fig. 6: MTDH–SND1 disruption and anti-PD-1 treatment synergistically inhibit metastatic breast cancer progression.
Fig. 7: C26-A6 treatment synergizes with anti-PD-1 therapy for metastatic breast cancer.
Fig. 8: MTDH expression negatively correlates with CD8+ T cell infiltration and PD-1 expression in patients with triple-negative breast cancer.

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Data availability

All RNA-sequencing data generated in this study have been deposited as a superseries at the NCBI Gene Expression Omnibus under accession code no. GSE174630. Further information and requests for resources and reagents should be directed to the corresponding author. All requests for raw and analyzed data and materials will be reviewed promptly by the corresponding author to verify whether the request is subject to any intellectual property or confidentiality obligations. Any data and materials that can be shared will be released via a material transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank G. Ren, W. Li, Z. Li, W. Lu and other laboratory members for technical support and helpful discussions. We thank W. Wang at the Genomics Core Facility of Princeton University for RNA-seq, C. DeCoste and K. Rittenbach at the Molecular Biology Flow Cytometry Resource Facility of Princeton University for the flow cytometry assays. We thank W. Zhang (University of Florida) for providing the E0771 cells. This research was supported by the Brewster Foundation and Ludwig Cancer Research and grants from the Breast Cancer Research Foundation, the National Institutes of Health (no. R01CA134519), Department of Defense Breast Cancer Research Program (no. BC151403), American Cancer Society and Susan G. Komen Foundation to Y.K. and postdoctoral fellowships from Susan G. Komen (no. PDF17332118) and NJCCR (no. DFHS15PPCO21) to M.S. This research was also supported by the Preclinical Imaging and Flow Cytometry Shared Resources of the Rutgers Cancer Institute of New Jersey (no. P30CA072720).

Author information

Authors and Affiliations

  1. Department of Molecular Biology, Princeton University, Princeton, NJ, USA

    Minhong Shen, Heath A. Smith, Yong Wei, Nicole Wang, Michelle Rowicki, Yong Tang, Xiang Hang, Liling Wan & Yibin Kang

  2. Key Laboratory of Breast Cancer in Shanghai, Department of Breast Surgery, Fudan University Shanghai Cancer Center, Shanghai, People’s Republic of China

    Yi-Zhou Jiang, Sheng Zhao, Songyang Wu & Zhi-Ming Shao

  3. Cancer Metabolism and Growth Program, The Cancer Institute of New Jersey, New Brunswick, NJ, USA

    Yibin Kang

  4. Ludwig Institute for Cancer Research Princeton Branch, Princeton, NJ, USA

    Yibin Kang

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Contributions

M.S. and H.A.S. designed and performed the experiments, analyzed the data and wrote the manuscript. Y.W. and L.W. generated the PyMT MTDH-WT/KO, Py8119-OVA cell lines and performed the RNA sequencing. N.W. and M.R. performed the in vitro tumor–immune cell coculture assays. Y.J., S.Z., S.W. and Z.S. collected the samples of patients with triple-negative breast cancer, performed the IHC staining and generated the survival plots. Y.T. performed the flow cytometry analysis. X.H. maintained the mouse strains and assisted with the animal experiments. Y.K. supervised the overall study, designed the experiments, analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Yibin Kang.

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Competing interests

Princeton has filed a disclosure on the findings based on this study. Y.K. and M.S. are named as co-inventors on the disclosure. Y.K. is a cofounder and chair of the Scientific Advisory Board of Firebrand Therapeutics, which is currently developing new therapeutics based on the targeting of MTDH–SND1 and has licensed relevant technologies from Princeton University. Y.K. is also a cofounder of Kayothera and a member of the Scientific Advisory Board for Cytocare and Vibrant Pharma Limited. The other authors declare no competing interests.

Additional information

Peer review information Nature Cancer thanks Daniel Ajona, Taosheng Chen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 MTDH depletion reshapes immune cell populations in tumors.

a, KO/PyMT cells were rescued with vector or wild type MTDH. The expression of MTDH was validated by western blot. b, Western blot analysis of endogenous MTDH knockdown in WT/PyMT cells. c, Western blot analysis of endogenous MTDH knockdown in E0771 cells with stable luciferase expression. d, 1 × 106 of E0771 cells with (shMTDH-1, -2) or without (shCtrl) MTDH knockdown were injected into female C57/BL6 mice via tail-vein. 6 weeks after injection, lungs were collected (left) and metastatic nodules were counted (right). n = 6 mice per group. Size bar, 5 mm. Data represent mean ± SEM. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. e, PyMT;UBC-CreERT+/-;Mtdhfl/fl mice with tumors established were treated with vehicle or Tamoxifen (Tmx) for five consecutive days. One week after the treatment, tumors were collected, and RNA was extracted for RNA sequencing. Gene sets that are significantly enriched in ranked gene list of Tmx treated versus control cells. n = 4 mice per group. f,g, Tumors from PyMT;UBC-CreERT+/-;Mtdhfl/fl mice treated with vehicle or Tmx were collected for immunohistochemistry (IHC) staining with indicated antibodies (f). The numbers of positive cells per field were quantified (g). Size bar, 50 μm. Data represent mean ± SEM. n = 8 fields from 4 mice in each group. Significance determined by two tailed Student’s t-test. h, PyMT;UBC-CreERT+/-;Mtdhfl/fl tumorspheres were treated with vehicle (Ctrl) or 4-Hydroxytamoxifen (4-OHT) (MTDH-KO). The spheres were then collected for RNA sequencing. Gene set enrichment analysis demonstrates the enrichment of the indicated gene sets. p and q values automatically determined by GSEA 3.0. The enrichment scores of the indicated signatures from tumorspheres in vitro or tumor samples in vivo (Fig. 1e) were presented (right). Numerical source data for d, g, and uncropped blots for a-c are provided.

Source data

Extended Data Fig. 2 CD8+ T cells depletion partially restores MTDH knockdown induced metastatic inhibition.

a, FVB females were treated with 125 μg/mouse of anti-CD8 antibody or isotype control for 3 days. Peripheral blood was collected for flow cytometry analysis at indicated days after treatment (top). % of CD8+ cells in CD45+ populations are shown. Anti-CD8 antibody treatment scheme that used for the in vivo experiments in this study (bottom). b, 1 × 106 E0771-shCtrl or shMTDH-1 (shMTDH hereafter) cells were injected into C57/BL6 females intravenously. The mice were subjected to isotype control or anti-CD4, anti-CD8 neutralizing antibodies treatment as in (a). 6 weeks after injection, the mice were euthanized, lungs were collected and fixed with Bouin’s solution. Representative lungs are shown and lung metastatic nodules were counted. n = 6 mice per group. Size bar, 5 mm. Data represent mean ± SEM. Significance determined by one-way ANOVA analysis with Sidak’s’s test for multiple comparisons. c, Kaplan-Meier survival curve of C57/BL6 female mice injected with 1 × 106 E0771-shMTDH that were treated with IgG or anti-CD8 neutralizing antibodies. n = 6 mice per group. Significance determined by Log-rank test. d, 5 × 105 -shCtrl or shMTDH E0771 cells were injected into the mammary fat pad of female C57BL6 mice, and the mice were treated with IgG or anti-CD8 neutralizing antibody as in (a). 6 weeks after injection, lungs were harvested and bioluminescent imaging (BLI) was performed to measure lung metastasis. Representative lungs (left), quantitative BLI signals (middle), and fold change of BLI signal between IgG and anti-CD8 groups (right) are shown. n = 16 mice per group. Data represent mean ± SEM. Significance determined by two tailed Student’s t-test. Numerical source data for b, c, d, are provided.

Source data

Extended Data Fig. 3 Characterization of in vitro tumor-immune cell co-culture system.

a, MHC-I (H-2Kd/H-2Dd) presentation in parental Py8119 cells were analyzed by flow cytometry. Isotype IgG served as negative control. b, Stable expression of Ovalbumin (OVA) in the resulted Py8119-OVA cells were confirmed with western blot. c, Surface presentation of OVA (H-2Kb-SIINFEKL) in parental Py8119 and Py8119-OVA cells with or without OT-1 splenocytes co-culture were analyzed with flow cytometry. d, Splenocytes isolated from OT-I mice were treated with PBS or 2 μg/ml of Ovalbumin peptide (OVAp257) for 2 hr. The cells were washed with PBS and plated in fresh media for another 24 hr followed by flow cytometry analysis. % of CD137+ or IFN-γ+ cells in live populations are shown. e, Splenocytes on the CD8 population were gated and the mean fluorescence intensity (MFI) of CD137 and IFN-γ were measured. n = 3 independent experiments. f, The indicated cells with endogenous Mtdh knockdown and vector (Vec) or wild type MTDH rescue were confirmed with western blot. g, The tumorigenesis ability of indicate cell lines were evaluated. Indicated cells were inoculated into the mammary fat pad of C57BL/6 female mice. Ten weeks after injection, lung metastatic nodules were counted. shCtrl, Py8119-shCtrl; KD, Py8119-shMTDH; shCtrl-OVA, Py8119-OVA-shCtrl; KD-OVA, Py8119-OVA-shMTDH; KD-OVA-Vec, Py8119-OVA-shMTDH rescued with vector; KD-OVA-MTDH, Py8119-OVA-shMTDH rescued with wild type MTDH. n = 5 mice per group. h, Py8119-OVA cells with/without endogenous Mtdh knockdown or with/without wild type MTDH rescued were employed for mammary fat pad injections. The injected OT-I female mice were treated with/without anti-CD8 neutralization antibody or IgG. Six weeks after treatment, lung metastasis was determined. n = 9 mice per group. i, Splenocytes co-cultured with indicated Py8119-OVA tumor cells (same as in Fig. 2b) for 24 hr were harvested for flow cytometry analysis. The expression of Granzyme B in CD8+ T cells were examined. MFI, Mean Fluorescence Intensity. n = 3 independent experiments. Data represent mean ± SEM. Significance determined by two tailed Student’s t-test (e,g), or one-way ANOVA analysis with Sidak’s’s test for multiple comparisons (h,i).Numerical source data for e, g, h, i, and uncropped blots for b, f are provided.

Source data

Extended Data Fig. 4 Mtdh acute loss enhances tumor antigen presentation.

a, PyMT;UBC-CreERT+/-;Mtdhfl/fl mice with tumors were treated with vehicle or 60 mg/kg of Tamoxifen (Tmx) for five consecutive days. One week after the treatment, tumors were collected, and RNA was extracted for RNA sequencing. Ingenuity pathway analysis shows the top five molecular and cellular functions of Mtdh acute loss up-regulated genes. p values automatically generated by QIAGEN Ingenuity Pathway Analysis (QIAGEN IPA). b, RNA samples from (a) were subjected to qRT-PCR test of indicated genes. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. c, Py8119-OVA-shCtrl or shMTDH tumor cells were co-cultured with OT-I splenocytes for 0 or 24 hr. Tumor cells were then collected and subjected to flow cytometry to determine the surface presentation of MHC-I. MFI, mean fluorescence intensity. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. Numerical source data for b, c are provided.

Source data

Extended Data Fig. 5 SND1 binds to Tap1/2 and promotes their degradation.

a, Endogenous SND1 knockdown in Py8119-OVA cells was confirmed with western blot. b, Tap1/2 mRNA levels in the indicated Py8119-OVA cells that were co-cultured with OT-I splenocytes for 0 or 24 hr was examined by qRT-PCR. n = 3 independent experiments. c, Indicated Py8119-OVA tumor cells co-cultured for 24 hr were treated with 10 μg/ml of actinomycin D for another 8 hr. RNA levels of Tap1/2 in tumor cells were determined by qRT-PCR. n = 3 independent experiments. d-g Py8119-OVA cells with SND1 (d) or MTDH (f) knockdown were subjected to RIP assay after 24 hr co-culture. The interaction between SND1 or MTDH and Tap1/2 was determined by PCR. Tap1/2 RNAs that bind to MTDH (e) or SND1 (g) were quantified and normalized to the pulled down MTDH and SND1 levels, respectively. n = 3 independent experiments. h, Electrophoretic mobility gel shift assay was performed with in vitro transcribed TAP1/2 mRNA incubated with PBS, recombinant MTDH and SND1 alone or in combination. Data represent mean ± SEM. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons (b,c,e), or two tailed Student’s t-test (g). Numerical source data for b, c, e, g,and uncropped images for a, d, f, h are provided.

Source data

Extended Data Fig. 6 SND1 inhibits antigen presentation and T-cell activation.

a-d, Py8119-OVA tumor cells (a,b) and OT-I splenocytes (c,d) after co-culture were collected to test OVA (H-2Kb-SIINFEKL) and MHC-I presentation, or CD137 and IFN-γ expression. MFI, mean fluorescence intensity; AU, arbitrary units. e, Media from (a) was employed for ELSA to test IFN-γ concentration and cytotoxicity assay. In panels from a-e, n = 3 independent experiments. f, PyMT tumor cells with MTDH KD or indicated rescues were injected into FVB females intravenously. Five weeks after injection, lung metastatic nodules were counted, and representative lungs were shown. n = 6 lungs per group. Data represent mean ± SEM. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons (a-e), or one-way ANOVA analysis with Sidak’s test for multiple comparisons (f). Numerical source data for a-f are provided.

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Extended Data Fig. 7 C26-A6 treatment elevates immune responses in tumors.

a, The correlation between all the gene sets that alters by Mtdh acute loss and C26-A6 treatment. b,c, Py8119-OVA cells co-cultured with OT-I splenocytes were treated with 200 μM of C26-A6 or same amount of vehicle. The binding between SND1 and Tap1/2 in tumor cells were determined by RIP assay (b). Tap1/2 RNAs that bind to SND1 were quantified and normalized to the pulled down SND1 levels (c). d, MHC-I presentation in Py8119-OVA cells with/without 200 μM of C26-A6 treatment in co-culture were determined by flow cytometry. MFI, mean fluorescence intensity; AU, arbitrary units. e,f, The expression of CD137 and IFN-γ in splenocytes co-cultured with Py8119-OVA were determined by flow cytometry upon 200 μM of C26-A6 or vehicle treatment. MFI, Mean Fluorescence Intensity; AU, arbitrary units. In panels from b-f, data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. Numerical source data for a, c-f and uncropped images for b are provided.

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Extended Data Fig. 8 MTDH-SND1 complex promotes immune evasion through Tap1/2.

a, Western blot analyzing to confirm the knock down of Tap1/2 in E0771 cells (E0771-OVA) stably expressing luciferase and OVA after lentiviral transduction of respective shRNAs. b-d, E0771-OVA cells with (shTap1/2) or without (shCtrl) Tap1/2 knockdown were co-cultured with OT-I splenocytes with the ratio of 1:10 (tumor cells:splenocytes). 24 hr after co-culture, Tumor cells (b,c) and splenocytes (d) were collected for examining OVA (H-2Kb-SIINFEKL), MHC-I, and CD137 expression, respectively. MFI, mean fluorescence intensity; AU, arbitrary units. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by one-way ANOVA analysis with Sidak’s test for multiple comparisons. e, The same co-culture experiment as in (b) were performed. The live tumor cells were indicated by luciferase signal. The percentage of live cells were determined as normalized to the non-co-culture control. Data represent mean ± SEM. n = 5 independent experiments. Significance determined by one-way ANOVA analysis with Sidak’s test for multiple comparisons. f, PresentER-Vector or PresentER-OVA (H-2Kb-SIINFEKL) system was stably expressed in Py8119 cells. The surface presentation of OVA (H-2Kb-SIINFEKL) was validated by flow cytometry analysis. g, Western blot analysis confirming the knock down of MTDH in Py8119 cells stably expressing luciferase and PresentER-OVA (Py8119-PresentER-OVA) after lentiviral transduction of respective shRNAs. h, Py8119-PresentER-OVA cells with (shMTDH) or without (shCtrl) MTDH knockdown were co-cultured with OT-I splenocytes with the ratio of 1:10 (tumor cells:splenocytes). 24 hr after co-culture, Tumor cells were collected for examining OVA (H-2Kb-SIINFEKL). Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. i, The same co-culture experiment as in (h) were performed. The live tumor cells were indicated by luciferase signal. The percentage of live cells were determined as normalized to the non-co-culture control. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. Numerical source data for b-e, h, i and uncropped blots for a and g are provided.

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Extended Data Fig. 9 MTDH-SND1 disruption and anti-PD-1 treatment synergistically enhance anti-tumor immune response.

a, OT-I splenocytes were co-cultured with Py8119-OVA cells with or without 200 μM of C26-A6 treatment for 24 hr. The expression of PD-1 in CD8+ T cells was examined by flow cytometry. % of CD8+PD-1+ in live populations are shown. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. b, Cells in (a) were gated on the CD8+ T cell population and the mean fluorescence intensity (MFI) of PD-1 expression was measured. AU, arbitrary units. Data represent mean ± SEM. n = 3 independent experiments. Significance determined by two tailed Student’s t-test. c, Schematic diagram of treatment. PyMT;UBC-CreERT+/-;Mtdhfl/fl females with tumors established were treated with Tmx and anti-PD-1 alone or in combination. Tmx, Tamoxifen, 60 mg/kg i.p. for 5 consecutive days; anti-PD-1, 200 μg/mouse i.p. injection, twice per week for the first week and then once per week after that. d, Primary tumors and lungs from experiment in Fig. 6c were fixed for CD8 IHC staining. Scale bar, 100 μm. Numerical source data for a and b are provided.

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Extended Data Fig. 10 C26-A6 combined with anti-PD-1 treatment reshapes the tumor immune microenvironment.

a,b, 100k PyMT tumor cells were orthotopically injected into the mammary glands of FVB females. The mice were randomized and divided into three groups when primary tumors were established, followed by vehicle, C26-A6, or C26-A6 + anti-PD-1 treatment. Six weeks after treatment, primary tumors and lung with metastatic lesions were collected for flow analysis with indicated antibodies. % of CD11b+F4/80+, Ly6GlowLy6Chigh, Ly6GhighLy6Clow, CD3−NK1.1+ in CD45+ population are shown. % of CD4+FOXP3+ in CD3+ population are shown, and % of GITR+LAG-3+ in CD8+ population are shown. Anti-PD-1, 200 μg/mouse i.p. injection, twice per week for the first week and then once per week after that; C26-A6, 15 mg/kg i.v. injection, 5 days per week. n = 6 mice per group. Data represent mean ± SEM. Significance determined by one-way ANOVA analysis with Dunnett’s test for multiple comparisons. c, Negative correlation between MTDH expression and CD8+ T cell infiltration or PD-1 expression in TNBC patients. Representative IHC images are shown in Fig. 8a. p-value by two-sided chi square test tests. Numerical source data for a and b are provided.

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Shen, M., Smith, H.A., Wei, Y. et al. Pharmacological disruption of the MTDH–SND1 complex enhances tumor antigen presentation and synergizes with anti-PD-1 therapy in metastatic breast cancer. Nat Cancer 3, 60–74 (2022). https://doi.org/10.1038/s43018-021-00280-y

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