Rac1 repression reverses chemoresistance by targeting tumor metabolism

ABSTRACT

Tumor metabolism is exemplified by the increased rate of glucose utilization, a biochemical signature of cancer cells. The enhanced glucose hydrolysis enabled by the augmentation of glycolytic flux and the pentose phosphate pathway (PPP) plays a pivotal role in the growth and survival of neoplastic cells. In a recent report, it has been shown that in human breast cancer the GTP binding protein, Rac1 enables resistance to therapy, particularly against the DNA-damaging therapeutics. Significantly, the findings demonstrate that Rac1-dependent chemoresistance involves the upregulation of glycolytic flux as well as PPP. Using multiple approaches, the study demonstrates that disruption of Rac1 activity sensitizes cancer cells to DNA-damaging agents. More importantly, the data uncover a previously unknown PPP regulatory role of Rac1 in breast cancer. Finally, the authors also show the effectiveness and the feasibility of in vivo targeting of Rac1 to enhance the chemosensitivity of breast cancer. This elegant report provokes scientific curiosity to expand our understanding of the intricacies of the role and regulation of Rac1 in cancer.

KEYWORDS:

• Rac1
• aldolase A
• pentose phosphate pathway (PPP)
• chemoresistance
• glucose metabolism
• triple-negative breast cancer (TNBC)

Deregulation of cellular bioenergetics is one of the hallmarks of cancer.² Among the energy-producing pathways, altered glucose metabolism is vital for cancer growth and development. Biochemically, the alteration involves rapid utilization of glucose via glycolysis rather than the mitochondrial oxidative phosphorylation (OxPhos). Recent research and several elegant reviews have significantly advanced our understanding of the role of tumor glycolysis and the molecular intricacies of its regulation.^3–6 Besides glycolysis, another mechanism that is strongly linked to glucose utilization and critical for cell survival is the pentose phosphate pathway (PPP) or hexose monophosphate shunt pathway that branches from the early steps of glycolysis. In fact, the product (glucose-6-phosphate, G6P) of the first committed step of glycolysis is the substrate for the first catalytic reaction of PPP by the enzyme, G6P-dehydrogenase (G6PDH). On the other hand, the metabolites of PPP (e.g. fructose-6-phosphate, glyceraldehyde-3-phosphate) serve as glycolytic intermediates and facilitate the maintenance of glycolytic flux. Thus, PPP and glycolysis support and to some extent regulate each other.

PPP generates pentose phosphates and nucleotides required for the nucleic acid biosynthesis, and also produces the reduced form of nicotinamide-adenine dinucleotide phosphate (NADPH), a major redox molecule. Thus, PPP plays an indispensable role in cell proliferation. Besides its function in the sustenance of growth and development, PPP also supports the survival of cancer cells exposed to therapeutics, particularly the DNA-damaging agents (e.g. Taxanes, platinum-based drugs) by promoting DNA-damage repair mechanisms. Noteworthy, such a resistance to therapy is a major challenge frequently encountered in the clinics and results in poor prognosis. Little is known about the molecular intricacies of PPP regulation that underlie chemoresistance. Li et al.,¹ in a recent report uncovered the link between GTP binding protein Rac1 and the PPP-related chemoresistance in breast cancer. Based on the microarray analysis of chemosensitive vs chemoresistant tumors the study finds that Rac1 (along with few other genes) is upregulated in triple-negative breast cancer (TNBC), and significantly correlated with the resistant phenotype.

Following the finding that Rac1 is associated with poor prognosis, Li et al., determined the relevance and regulation of Rac1 in chemoresistance. Using diverse approaches like Rac1 depletion (RNAi approach) or overexpression in chemoresistant and chemosensitive breast cancer cells, the report documents that Rac1 confers chemoresistance, especially to DNA-damaging therapeutics. Next, the study focused on understanding the mechanism underlying Rac1- mediated resistance. Quantitative analysis of various metabolic intermediates and the expression of specific enzymes revealed that Rac1 upregulates the glycolytic enzyme, aldolase, and the enzymes of non-oxidative PPP, ribulose 5-phosphate isomerase (RPI), and transketolase (TKTL). Accordingly, - Rac1 depletion reduced the rate of glucose uptake with a concomitant decrease in the glycolytic flux and PPP. Further investigation on therapeutic targeting demonstrated the relevance and feasibility of in vivo inhibition of Rac1 to sensitize breast cancer to DNA-damaging therapeutics. Thus, Li et al., document a previously unknown metabolic-regulatory role of Rac1 which is linked to the chemoresistance in breast cancer.

Emerging reports underscore the significance of PPP in cancer cell proliferation and survival. While the PPP-metabolite pentose phosphate is critical for nucleic acid biosynthesis, the NADPH generated in the process is essential for fatty acid synthesis, as well as the mitigation of cellular oxidative stress.⁷ Results of several preclinical studies have indicated that PPP-inhibition disrupts cancer growth accentuating the therapeutic potential of anti-PPP, anticancer strategy. For example, inhibition of the PPP enzyme, 6-phosphogluconate dehydrogenase (6PGDH) sensitizes breast cancer to chemotherapy.⁸ 6PGDH downregulation disrupts de novo lipid biosynthesis by active-AMPK-mediated inhibition of acetyl CoA carboxylase 1 (ACC1). Similarly, in lung cancer, the enzyme, 4-hydroxyphenyl pyruvate dioxygenase (4HPD) transcriptionally upregulates G6PDH which catalyzes the first oxidative step of PPP. Accordingly, inhibition of either 4HPD or its molecular target G6PDH is sufficient to abrogate PPP and the related signaling pathway leading to the disruption of nucleic acid biosynthesis, augmentation of oxidative stress to sensitize cancer cells for therapy.⁹

Next, as NADPH plays an indispensable role in the maintenance of intracellular homeostasis, interventional strategies to deplete PPP-derived NADPH pool remains an attractive target in anticancer research. Cancer cells overexpress the amino acid transporter, SLC7A11, which is required for the import of cystine. Owing to the upregulation of SLC7A11, the intracellular accumulation of cystine increases in cancer cells. Noteworthy, unless the cystine is reduced and converted into the cysteine, it will be toxic to cancer cells. The reductive process of cystine to cysteine utilizes NADPH, one of the by-products of PPP. Thus, any disruption of PPP that diminishes NADPH pool is likely to increase cystine-overload causing cell death or at least may increase the vulnerability to therapeutic interference.¹⁰ Similarly, in cancer cells with isocitrate dehydrogenase 1 (IDH1) mutation, the rate of PPP is increased due to 2-hydroxyglutarate (2HG) synthesis.¹¹ Since 2HG synthesis consumes considerable NADPH generated by PPP, in order to maintain sufficient NADPH pool required for other essential functions (e.g. redox balance), PPP in general is upregulated in IDH1 mutant cells. In other words, IDH1 mutant cancer cell’s dependency on augmented PPP reveals a metabolic vulnerability that may be exploited by oxidative stress-inducing therapies. Thus, PPP is considered as a potential target for cancer treatment. The current report documents a molecular approach using Rac1-inhibition strategy to disrupt PPP, and sensitize breast cancer for therapy.

Data from earlier studies also indicate that Rac1 inhibition reverses chemoresistance by disrupting tumor glycolysis.¹² However, little is known about the underlying mechanism. From a metabolic regulation perspective, Rac1 activates the hypoxia-inducible factor (HIF) 1, a master regulator of several proteins including the enzymes of the glycolytic pathway.¹³ Consequently, downregulation of Rac1 reduces HIF1α level with a concomitant decrease in glycolytic enzymes and overall rate of glycolysis.¹⁴ Specifically, HIF1α upregulates aldolase A, the glycolytic enzyme that catalyzes the conversion of fructose 1,6-bisphosphate (F-1,6BP) into dihydroxyacetone phosphate (DHA) and glyceraldehyde-3-phosphate (G3P).^15,16 Hence, the inhibition of HIF1α downregulates aldolase and disrupts specific glycolytic reaction.¹⁷ Intriguingly, Rac1 also regulates aldolase expression, and it remains to be known if it involves a Rac1-HIF1α axis or independent of HIF1α activity.

Functionally, the tumorigenic capacity of Rac1 is known in multiple types of cancer. However, there is a lack of homogeneity on the role of Rac1 in cancer growth or drug sensitivity. In liver cancer, Rac1 deletion significantly reduces tumor growth, in agreement with its tumorigenic function.¹⁸ Similarly, the association of Rac1 in the development of chemoresistant phenotype is also evident in esophageal cancer.¹² Conversely, in lung cancer, induction of DNA-damage and growth arrest has been found to be associated with increased Rac1 activity, indicating an anticancer role of Rac1.¹⁹ Also, in leukemic cells, Rac1 inhibition but not its upregulation confers resistance to doxorubicin, indicating a proapoptotic role for Rac1.²⁰ Taken together, these results suggest that Rac1 role in drug sensitivity or resistance may be differentially regulated and may vary among different cancers.

As for the regulation of Rac1in TNBC, the protein Tuftelin 1 (TUFT1) is significantly associated with the disease progression, and correlates with Rac1 expression. Upregulation of TUFT1 increases chemoresistance resulting in poor prognosis, the phenotype exhibited by Rac1 as well. Importantly, the inhibition of TUFT1 induces apoptosis by downregulation of Rac1.²¹ These findings imply a TUFT1-dependent regulation of Rac1 in TNBC. Next, analogous to the finding that PPP-related enzymes (RPI, TKTL) are regulated by Rac1,¹ the occurrence of an inverse regulation is also known. In cervical cancer, downregulation of the PPP-enzyme, 6PGDH results in decreased Rac1 activity and enhances chemosensitivity.²² Nevertheless, Rac1 downregulation of PPP and/or PPP (enzyme)-mediated inhibition of Rac1, both abrogate therapeutic resistance in cancer.

Metabolic reprogramming, particularly the PPP facilitates the development of resistance to therapeutics that rely on DNA-damage-induced apoptosis (e.g. cisplatin).²³ The PPP enzymes such as G6PD, 6PGDH, 6-phosphogluconolactonase [6PGL], and TKTL are among the potential targets that showed promising results in preclinical studies.^23,24 Little is known about the regulation of PPP in cancer cells, as recent data indicate a role for myriad of proteins known for diverse cellular functions to be associated with PPP regulation. For instance, the upregulation of PPP by suppression of the glycolytic enzyme, phosphofructokinase 2 (PFK2) isoform, has been indicated in DNA-damage repair response and recovery of cancer cells. Notably, p53 which is implied in cell cycle arrest and apoptosis is involved in the suppression of PFK2 isoform, to upregulate PPP.²⁵ Another protein involved in the cell division and cell cycle, polo-like kinase 1 (PLK1) is known to accelerate PPP flux to facilitate the progression of mitosis. Specifically, PLK1 phosphorylates G6PD which in turn augments the rate of PPP. Experimental manipulation confirmed that PLK1-interaction with G6PD is necessary for cell cycle progression and cancer growth.²⁶ Collectively, current literature indicate that complex mechanisms or pathways may impact the regulation of PPP depending upon the requirements of cancer cells. In summary, future studies on the molecular intricacies that regulate PPP in individual tumor types and at various stages of progression may enable us to develop an effective and translatable strategy to achieve personalized cancer therapy.