Multiomics integrative analysis reveals antagonistic roles of CBX2 and CBX7 in metabolic reprogramming of breast cancer

  1. Mohammad Askandar Iqbal
  2. Shumaila Siddiqui
  3. Asad Ur Rehman
  4. Farid Ahmad Siddiqui
  5. Prithvi Singh
  6. Bhupender Kumar
  7. Daman Saluja

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Abstract

Striking similarity exists between metabolic changes associated with embryogenesis and tumorigenesis. Chromobox proteins-CBX2/4/6/7/8, core components of canonical polycomb repressor complex 1 (cPRC1), play essential roles in embryonic development and aberrantly expressed in breast cancer. Understanding how altered CBX expression relates to metabolic reprogramming in breast cancer may reveal vulnerabilities of therapeutic pertinence. Using transcriptomic and metabolomic data from breast cancer patients (N>3000 combined), we performed pathway-based analysis and identified outstanding roles of CBX2 and CBX7 in positive and negative regulation of glucose metabolism, respectively. Genetic ablation experiments validated the contrasting roles of two isoforms in cancer metabolism and cell growth. Furthermore, we provide evidence for the role of mTORC1 signaling in mediating contrary effects of CBX2 and CBX7 on breast cancer metabolism. Underpinning the biological significance of metabolic roles, CBX2 and CBX7 were found to be the most up- and down-regulated isoforms, respectively, in breast tumors compared to normal tissues. Moreover, CBX2 and CBX7 expression (not other isoforms) correlated strongly, but oppositely, with breast tumor subtype aggressiveness and the proliferation markers. Consistently, genomic data also showed higher amplification frequency of CBX2, not CBX7, in breast tumors. Highlighting the clinical significance of findings, disease-specific survival and drug sensitivity analysis revealed that CBX2 and CBX7 predicted patient outcome and sensitivity to FDA-approved clinical drugs. In summary, this work identifies novel cross-talk between CBX2/7 and breast tumor metabolism, and the results presented may have implications in strategies targeting breast cancer.

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  1. Version published to 10.1101/2020.08.06.239129v2 on bioRxiv
  2. eLife

    ###Reviewer #3

    This manuscript describes analysis and experiments designed to implicate CBX2 and CBX7 in breast carcinogenesis. Naturally, the analysis of existing data provides only correlative measures, and some of these are likely insignificant and driven by outliers (see specific points below). The experimental validation is done in two cell lines with a single siRNA, and data showing successful targeting of siRNA is lacking. The authors also claim direct regulation of mTORC by CBX2 and CBX7, but the evidence provided is weak. Overall the results are suggestive but do not provide conclusive evidence justifying the conclusions.

    Specific Points:

    The expression of CBX2/CBX7 correlates with breast cancer subtype, so all the predictive power may be in the subtype of cancer. Is there evidence that once standard prognostic methods are applied, CBX2 and/or CBX7 expression levels add to prediction? If not, it is not clear that these are drivers and not simply correlative markers of disease status.

    Figure 2 should include CBX2, CBX7, and other CBX RNA and protein levels to show that targeting was effective and specific. Multiple siRNAs should be used to demonstrate that it is not an off-target effect.

    Figure 3 correlations are extremely weak. Significance is driven by the large number of data points and not by correlation, and likely it is also driven by the few outliers on the left in each figure. If these are removed correlation is likely close to zero.

  3. eLife

    ###Reviewer #2

    Saluja and colleagues present a study examining the contribution of chromobox-family of proteins, specifically to CBX2/7, on metabolic reprogramming of breast cancer cells. Notably, little is known regarding CBX2/7's activity in metabolism. The manuscript is well written and clearly presented. The major findings are that CBX2 and 7 are related to metabolic reprogramming and have inverse roles in regulating anerobic glycolysis, respectively. Through mining of several large datasets (TCGA/METABRIC), investigators demonstrate that amplification and upregulation of CBX2 correlates to more aggressive tumors and correlates to increased mTORC signaling. Authors directly demonstrate that siRNA knockdown of CBX2 leads to loss of glucose uptake and a reduction in ATP production. Conversely, loss of CBX7 increased glucose uptake, increased ATP production, promoted an increase in cell number, and pS6 phosphorylation. There is a significant need to better define the contribution of CBX2 and CBX7 in breast cancer, which will shed light on breast cancer progression, metabolic reprogramming, and therapeutic response. The strengths of the study included the use of large, well-annotated datasets and a novel area of cross-talk between epigenetics and metabolism. However, there are concerns detailed below that need to be addressed:

    Major:

    1. Most of the research presented is correlative studies with little mechanistic insight. CBX2 and CBX7 are members of the polycomb repressor complex 1 (PRC1). Are the CBX2 and CBX7 expression mutually exclusive? Related to figure 3, what is the mechanism of action that loss of CBX2 expression and decreases mTORC signaling? CBX2 and CBX7 proteins are not likely functioning alone. In CBX2High cell lines authors should investigate the impact of a PRC1 inhibitor in the context of anaerobic glycolysis to assess whether the CBX2 is functioning independent of PRC1. Also, the discussion regarding the interplay between PRC1, PRC2, and metabolism should be included.

    2. The MTT and Cell titer glo therapeutic sensitivity assays need to be repeated using a non-metabolic readout. The major conclusion of the study is that CBX2 and CBX7 promote metabolic reprogramming thus using metabolic outputs (Cell Titer Glo - ATP production and MTT - mitochondrial respiration) for the chemotherapy assays are flawed.

    3. Only two cell lines examined (MCF7 [ER/PR positive] and MDA-MB-231 [triple negative]), which is a study limitation. Why were these cell lines selected? Also, only pooled siRNA for both CBX2 and CBX7 were used, thus only loss-of-function responses are evaluated. Does overexpression of CBX2 in a CBX2-low cell line exacerbate anaerobic glycolysis and conversely does CBX7 overexpression in CBX7-low inhibit anaerobic glycolysis?

    4. Based on figure 6, the CBX2high lines are less responsive to Rapamycin suggesting that the cells are not dependent on CBX2-mediated upregulation of mTORC. Temsirolimus was also not detected as being significant, further highlighting that CBX2-activity on mTORC is not a critical pathway. Also, given the antagonistic effect of CBX7, what are the therapeutic vulnerabilities conveyed in CBX7high?

    5. The survival curves demonstrated in Figure 5 show a substantial difference between TCGA and Metabric data, what is the possible explanation?

  4. eLife

    ###Reviewer #1

    The manuscript entitled "CBX2 and CBX7 antagonistically regulate metabolic reprogramming in breast cancer" analyzed multi-omics data of breast cancer mainly from METABRIC and TCGA with the focus on the chromobox family member genes (CBXs). Authors showed the association of CBX2 and CBX7 expression levels with glycolysis in tumors and the mTOR signaling, especially the levels of phosphorylation of S6 protein in tumors. Knockdown of CBX2 and CBX7 in two breast cancer cell lines showed opposite effects on glycolysis, cell viability and growth. Previous studies reported that CBX2 and CBX7 have oncogenic and tumor-suppressive roles in breast cancers. Results from this study showed their involvement in regulation of glycolysis, as well as their association with the prognosis of disease-specific survival of breast cancers. While some of the findings about CBX2 and CBX7 are interesting, most of the results showed association and provided limited insights about how CBX2 and CBX7 regulates glycolysis and their contribution in breast cancer.

    Specific comments:

    1. The authors need to provide detailed methods of analysis, including glycolysis deregulation score, where to obtain the DNA methylation levels, etc.

    2. It is uncertain that it is acceptable practice to base/categorize breast cancer aggressiveness according to different subtypes (from LumA, LumB, Her2 to Basal) as shown in Figure 1D, Figure 4C, 4F.

    3. 2-DG experiments were only performed in MDA-MB-231 and MCF-con cells but not cells with CBX knockdown (Fig S3). It is therefore unclear whether the changes of cell viability, proliferation by CBX knockdown are due to the metabolic changes (Figure 2).

    4. Figure 3 showed the effects of CBX on pS6 levels in breast tumors. However, it is unclear whether this change contributes to the role of CBX2, CBX7 in glycolysis. The statement on page 6, line 1 "CBX2 and CBX7 exert their effects on breast cancer metabolism via modulation of mTORC1 signaling" is not established and has no data to support.

    5. Figure 5, since the % of CBX2 high/low and CBX7 high/low differ in different subtypes of breast cancers, it is suggested to analyze the association of CBX2, CBX7 expression with prognosis in different subtypes.

    6. Figure 6, please discuss why CBX2 high cells which supposedly have high mTOR activity showed higher resistance towards Rapamycin compared to CBX2 low cells. Also, whether CBX7 showed opposite effects of drug sensitivity towards the same group of compounds.

  5. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    The manuscript has been reviewed by three experts in the field, including an expert in metabolism, one in breast cancer and one in bioinformatics. There are concerns that much of the data are correlative as opposed to mechanistic, and that the material thus falls short of increasing insights into the role of CBX2/7 in breast cancer. There is concern that the cell viability assays are actually readouts of metabolism, and that viability assays should be repeated using a non-metabolic readout such as trypan blue or calcein/EtBr stain. There are concerns about the possibility that the expression of CBX2 and 7 as markers of breast cancer subtype are actually driving the correlations seen. And there are concerns that only two cell lines are analyzed, and only a single siRNA. It is suggested that performing the metabolism assays in the presence of knockdown for CBX would better support the premise that there is a correlation between metabolism and proliferation, and that these are together regulated by CBX proteins. Finally, one of the reviewers requests more detail in the methods.

  6. Version published to 10.1101/2020.08.06.239129v1 on bioRxiv