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Metabolic enzyme LDHA activates Rac1 GTPase as a noncanonical mechanism to promote cancer

Nature Metabolism volume 4, pages 1830–1846 (2022)Cite this article

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Abstract

The glycolytic enzyme lactate dehydrogenase A (LDHA) is frequently overexpressed in cancer, which promotes glycolysis and cancer. The oncogenic effect of LDHA has been attributed to its glycolytic enzyme activity. Here we report an unexpected noncanonical oncogenic mechanism of LDHA; LDHA activates small GTPase Rac1 to promote cancer independently of its glycolytic enzyme activity. Mechanistically, LDHA interacts with the active form of Rac1, Rac1–GTP, to inhibit Rac1–GTP interaction with its negative regulator, GTPase-activating proteins, leading to Rac1 activation in cancer cells and mouse tissues. In clinical breast cancer specimens, LDHA overexpression is associated with higher Rac1 activity. Rac1 inhibition suppresses the oncogenic effect of LDHA. Combination inhibition of LDHA enzyme activity and Rac1 activity by small-molecule inhibitors displays a synergistic inhibitory effect on breast cancers with LDHA overexpression. These results reveal a critical oncogenic mechanism of LDHA and suggest a promising therapeutic strategy for breast cancers with LDHA overexpression.

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Fig. 1: LDHA directly interacts with Rac1.
Fig. 2: LDHA activates Rac1 in breast cancer cells and mouse mammary tissues.
Fig. 3: LDHA binds to Rac1–GTP and inhibits its binding with Rac1 GAPs to activate Rac1.
Fig. 4: LDHA activates Rac1 to promote colony formation, migration and invasion of breast cancer cells.
Fig. 5: LDHA activates Rac1 to promote growth and metastasis of breast tumors.
Fig. 6: LDHA and Rac1 small-molecule inhibitors display a synergistic inhibitory effect on colony formation, migration and invasion of breast cancer cells.
Fig. 7: LDHA and Rac1 small-molecule inhibitors display a synergistic inhibitory effect on primary and metastatic breast tumors.

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Data availability

Publicly available datasets used in this study are TGCA (https://portal.gdc.cancer.gov/), Oncomine (https://www.oncomine.com/) and Cancer Cell Line Encyclopedia (https://sites.broadinstitute.org/ccle/). All data supporting the present study are available within the article and supplementary information files. Source data are provided with this paper.

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Acknowledgements

LC–MS/MS proteomic analysis was performed at the Biological Mass Spectrometry facility of Rutgers University. This work was supported in part by grants from the National Institutes of Health (R01CA227912 and R01CA214746 to Z.F., as well as R01CA203965 and R01CA260837 to W.H.) and Congressionally Directed Medical Research Programs (CA214746 to Z.F.). T.Z. and C.C. were supported by the postdoctoral fellowship from New Jersey Commission on Cancer Research.

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Authors and Affiliations

  1. Department of Radiation Oncology, Rutgers Cancer Institute of New Jersey, Rutgers-The State University of New Jersey, New Brunswick, NJ, USA

    Juan Liu, Cen Zhang, Tianliang Zhang, Chun-Yuan Chang, Jianming Wang, Ludvinna Bazile, Bruce G. Haffty, Wenwei Hu & Zhaohui Feng

  2. Department of Biological Sciences, Rutgers-The State University of New Jersey, Newark, NJ, USA

    Lanjing Zhang

  3. Department of Pathology, Princeton Medical Center, Plainsboro, NJ, USA

    Lanjing Zhang

  4. Department of Pharmacology, Rutgers-The State University of New Jersey, Piscataway, NJ, USA

    Wenwei Hu & Zhaohui Feng

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Contributions

J.L., C.Z., T.Z., C.C., J.W. and L.B. performed the experiments and analyzed data. L.Z. and B.G.H. analyzed data and contributed important materials. W.H. and Z.F. conceived and supervised the study. J.L., W.H. and Z.F. wrote the manuscript.

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Correspondence to Wenwei Hu or Zhaohui Feng.

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Nature Metabolism thanks William J. Muller, Taro Hitosugi and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Alfredo Giménez-Cassina, in collaboration with the Nature Metabolism team.

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Extended data

Extended Data Fig. 1 LDHA overexpression and its association with clinical outcomes in different subtypes of breast cancers classified by the status of ER, PR or HER2.

a-c, The increased LDHA mRNA levels in different subtypes of breast cancers compared with matched adjacent non-tumor breast tissues. The data were obtained from TCGA and the P-value was analyzed by two-tailed paired Student’s t-test. d-f, High LDHA mRNA expression is associated with poor relapse-free survival in patients with different subtypes of breast cancers. The data were obtained from Kaplan-Meier plotter (http://kmplot.com) and analyzed by the log-rank (Mantel–Cox) test. ER: estrogen receptor; PR: progesterone receptor.

Extended Data Fig. 2 LDHA displayed a much weaker interaction with Rac1-T35S mutant compared with WT Rac1 in cells.

Hs578T cells with ectopic expression of LDHA-Flag and WT Myc-Rac1, Myc-Rac1-T17N, or Myc-Rac1-T35S were employed for co-IP assays followed by western-blot assays. Data represent three repeats with similar results.

Source data

Extended Data Fig. 3 The dominant negative Rac1-T17N mutant reduces the promoting effect of WT and L4 LDHA on colony formation, migration and invasion of breast cancer cells.

a-c, Hs578T and SK-BR3 cells with ectopic expression of WT or L4 LDHA were transduced with control or Rac1-T17N expression vectors for colony formation (a), migration (b) and invasion (c) assays. Data represent mean ± s.d. (n = 6 independent experiments), two-way ANOVA followed by Tukey’s or Bonferroni’s test. *: P < 0.0001.

Source data

Extended Data Fig. 4 LDHA but not Rac1 is localized in invadopodia in breast cancer cell lines.

HS578T and MDA-MB231 cells seeded on gelatin-coated coverslips were labeled with anti-LDHA or Rac1 in far-red, anti-Tks5 in green and phalloidin (to stain F-actin) in red. The co-localization of Tks5 and F-actin in a punctate manner in cells was used as an indication of invadopodia formation. Arrows indicate invadopodia in cells. Scale bars: 20 μm. Data represent three repeats with similar results.

Extended Data Fig. 5 The potential role of different Rho family proteins in mediating the oncogenic effect of LDHA in breast cancer cells.

a, The interaction between LDHA with different Rho family proteins analyzed by co-IP assays. Hs578T cells expressing LDHA-Flag and Myc-Rac1, Myc-Cdc42, Myc-Rac3, or Myc-RhoA were used for co-IP and western-blot assays. b, LDHA activated Rac3 and Cdc42, but at a much less extent compared with its effect on Rac1. Hs578T and SK-BR3 cells expressing LDHA-Flag were used for PAK-PBD pull-down assays to measure the levels of GTP-bound Rac1, Rac3 and Cdc42. Left panels: represented results. Right panels: relative Rac1, Rac3 and Cdc42 activities analyzed by comparing the levels of GTP-bound Rac1, Rac3 and Cdc42 to the levels of total Rac1, Rac3 and Cdc42 proteins, respectively, in cells. Data represent n = 3 independent experiments, two-tailed unpaired Student’s t-test. c, Knockdown of Rac3 or Cdc42 displayed a much less pronounced effect on colony formation, migration and invasion of breast cancer cells with WT or L4 LDHA expression compared with Rac1 knockdown. Data represent mean ± s.d. (n = 6 independent experiments), two-way ANOVA followed by Tukey’s test or Dunnett’s test. d, Knockdown of Rac3 and Cdc42 in cells was confirmed by Taqman real-time PCR assays. Their mRNA levels were normalized with Actin. Data represent n = 3 independent experiments, one-way ANOVA followed by Dunnett’s test. *: P < 0.0001.

Source data

Extended Data Fig. 6 The relative expression levels of Rac1, Rac3 and Cdc42 in breast cancer cells.

Rac1 has a much higher expression level than Rac3 and Cdc42 in majority of breast cancer cell lines, including cell lines used in this study (labeled in red) as shown by the RNA-Seq data from Cancer Cell Line Encyclopedia (CCLE; https://sites.broadinstitute.org/ccle/).

Extended Data Fig. 7 LDHA and Rac1 small-molecule inhibitors display a much more pronounced inhibitory effect on colony formation, migration and invasion of breast cancer cells.

a, The combination treatment displayed a much more pronounced inhibitory effect on colony formation of breast cancer cells. BT-549, MCF7 and ZR-75-1 cells were treated with indicated concentrations of FX11 and/or NSC23766 for 4 days before colony formation assays. Combo: FX11 + NSC23766. b, c, The combination treatment displayed a much more pronounced inhibitory effect on migration (b) and invasion (c) of breast cancer cells than the single inhibitor treatment as analyzed by Transwell assays. In b, c, BT-549, MCF7 and ZR-75-1 cells were treated with the indicated concentrations of FX11 and/or NSC23766 for 24 h. NSC: NSC23766. Data represent mean ± s.d. (n = 6 independent experiments), one-way ANOVA followed by Tukey’s test. *: P < 0.0001.

Source data

Extended Data Fig. 8 The effect of small-molecule inhibitor treatments on the body weights of tumor-bearing mice.

a, Small-molecule inhibitor treatments did not significantly affect the body weights of female nude mice bearing orthotopic tumors formed by WT Hs578T cells. b, Small-molecule inhibitor treatments did not significantly affect the body weights of female BALB/c mice bearing orthotopic tumors formed by 4T1 cells transduced with the control lentiviral shRNA vector. Mice were treated with FX11 (1 mg/kg/day; i.p.; once/day) and/or NSC23766 (1.5 mg/kg/day, i.p.; once/day), or vehicle (−). Mice with Hs578T tumors were treated for 3 weeks and mice with 4T1 tumors were treated for 2 weeks. The body weights of mice were measured and recorded at the days indicated. Data represent mean ± s.d. n=8 mice/group. Statistical differences were determined by two-way ANOVA followed by Tukey’s test.

Source data

Extended Data Fig. 9 The effect of FX11 and/or NSC23766 on the growth and lung metastasis of mammary tumors in MMTV-PyMT mice.

a, The effects of FX11 and/or NSC23766 on the growth and metastasis of mammary tumors in 10-week-old MMTV-PyMT mice. The 10-week-old female MMTV-PyMT mice that developed late-stage tumors were treated with FX11 (1 mg/kg/day; i.p.; once/day) and/or NSC23766 (1.5 mg/kg/day, i.p.; once/day) for 3 weeks before they were sacrificed for analysis. Data represent mean ± s.d.; n=6 mice/group. One-way ANOVA followed by Student’s t-test. Con: vehicle; NSC: NSC23766; combo: FX11 + NSC. b, The interaction between endogenous LDHA and Rac1 in both non-tumor and tumor mammary tissues of MMTV-PyMT mice detected by co-IP and western-blot assays. Non-tumor mammary tissues were obtained from 5-week-old MMTV-PyMT mice and mammary tumor tissues were obtained from 10-week-old MMTV-PyMT mice. Normal mammary tissues from 5-week-old LDHA-deficient mice (the R26-Cre-ERT2, LDHAflox/flox mice treated with Tamoxifen to delete LDHA as presented in Fig. 2h) were used as negative controls for assays. NC: negative control.

Source data

Extended Data Fig. 10 LDHA expression is not linked to any specific subtypes of breast cancer.

LDHA protein levels in two breast cancer TMAs, including TMA-RCINJ (a-c; n=200) and TMA-BR2082a (d-f; n=120), were analyzed by IHC staining and were compared in different cancer subtypes classified by the status of ER (a, d), PR (b, e), or HER2 (c, f). Statistical studies were performed by using χ2 test. The TMA-BR2161 does not have information on the status of ER, PR or HER2.

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Supplementary Information

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Statistical source data for Extended Data Fig. 10.

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Liu, J., Zhang, C., Zhang, T. et al. Metabolic enzyme LDHA activates Rac1 GTPase as a noncanonical mechanism to promote cancer. Nat Metab 4, 1830–1846 (2022). https://doi.org/10.1038/s42255-022-00708-4

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