HER2 is a member of the epidermal growth factor (EGF) receptor family comprising EGFR/ErbB1, HER2/ErbB2, HER3/ErbB3, and HER4/ErbB4. EGF receptor family proteins encode for transmembrane receptors consisting of the extracellular ligand binding, lipophilic transmembrane, and cytoplasmic tyrosine kinase domains. Ligand binding to EGF receptors activates their hetero- or homodimerization whereby one EGF receptor will phosphorylate the other on a tyrosine, allowing cytoplasmic signaling complexes to bind to the EGFR dimer. This stimulates signal transduction cascades such as the PI3K-Akt-mTOR and MAPK pro-survival pathways ^[7][12][14,19] (reviewed in ^[13][20]). However, the proto-oncogene Neu/HER2 (HER2) has no known natural ligands ^[14][21]; thus, to elicit downstream signaling, HER2 preferentially dimerizes with EGF receptors that subsequently results in a more potent signaling response. Due to its role in activating signaling pathways that stimulate cell proliferation and survival, HER2 overexpression in breast tumor cells promotes tumor growth and increases the percentage of cells in S phase, aneuploidy, and migration of cancer cells into the lymph nodes ^[15][22] (reviewed in ^[16][23]). Spontaneous homodimerization of HER2 is more prominent in HER2-positive breast cancer, shifting the balance of the EGR receptor dimers towards a greater ratio of homodimers ^[12][17][19,24]. Following dimerization, EGF receptors undergo endocytosis and are either recycled back to the cell surface or targeted for proteasomal degradation by ubiquitin ligase Cbl ^[18][25]. Dimerization stimulates EGFR/ErbB1 phosphorylation (Y1045) and recruitment of Cbl ^[18][19][25,26]; however, in heterodimers with ErbB2, Cbl recruitment is impeded. Although ErbB2 is phosphorylated (Y1112), Cbl recruitment is inefficient, and EGF receptors are recycled back to the cell surface ^[20][27].

Enhanced activity of mTOR is associated with HER2-overexpressing breast cancers ^[50][51][77,78]. The mTOR protein contains five domains: (i) the Huntington, EF3A, ATM, TOR (HEAT) repeats, (ii) the helical Frap, ATM, TRRAP (FAT) domain, (iii) FKBP–rapamycin complex binding (FRB), (iv) the kinase domain, and (v) FAT C-terminal (FATC). mTOR is the catalytic subunit in two protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 is composed of five components: mTOR, regulatory-associated protein of mTOR (raptor), mammalian lethal with Sec13 protein 8 (mLST8 or GβL), proline-rich Akt substrate 40 kDa (PRAS40), and DEP-domain-containing mTOR-interacting protein (deptor). Like mTORC1, mTORC2 also contains mTOR, mLST8, and deptor, but has the rapamycin-insensitive companion of mTOR (rictor), mammalian stress-activated protein kinase interacting protein (mSIN1), and protein observed with Rictor-1 (Protor-1) subunits ^[52][79]. Raptor mediates mTORC1 binding to the substrates for phosphorylation ^[53][80]. Rictor is unrelated to raptor but facilitates the phosphorylation of various substrates of mTORC2 ^[54][81].

Serine-threonine kinase LKB1 (STK11, also referred to as Liver Kinase 1), is a tumor suppressor that negatively regulates mTORC1 activity. LKB1 functions within a heterotrimer, containing the pseudokinase STRAD and the adaptor protein MO25 ^[55][56][57][82,83,84], that activates AMPK by phosphorylation on T172^[58] (Figure 1; [85]). AMPK, also a serine-threonine kinase, is a metabolic sensor and negative regulator of the mTOR signaling pathways. The AMPK protein is composed of the catalytic α subunit and the regulatory β and γ subunits. AMPK activation is induced by energy-stressed conditions in which intracellular AMP levels are elevated. AMP activates AMPK by binding to the γ subunit and subsequently targeting it for phosphorylation by LKB1 on T172 ^[59][86]. AMPK activates the tuberous sclerosis complex 1 and 2 (TSC1/2) by phosphorylation of T1227 and S1345 on TSC2 ^[60][87]. Activated TSC1/2 acts as a GTPase activating protein (GAP) that inhibits the small GTPase Ras Homolog Enriched in Brain (Rheb), which is involved in mTORC1 activation ^[61][88]. In addition, AMPK can directly inhibit mTORC1 by phosphorylation of raptor on S722 and S792 ^[62][89].

The PI3K-Akt pathway is an important regulator of the mTOR pathway. The Akt protein consists of three main domains: the N-terminal pleckstrin homology (PH) domain, the central kinase catalytic (CAT) domain, and the C-terminal extension (EXT) region. The PH domain mediates the protein–protein and protein–lipid interactions of Akt. The CAT domain mediates enzymatic activity and contains T308, a phosphoinositide-dependent kinase 1 (PDK1)-dependent phosphorylation site necessary for Akt activation ^[63][90]. The EXT region contains S473 phosphorylation for full Akt activation (reviewed in ^[64][91]). Akt enables mTOR activity by inactivating TSC1/2 through the phosphorylation of TSC2 on several residues, hence enabling Rheb-GTPase to activate mTORC1^[65] (Figure 1; [92]). Akt can also directly activate mTORC1 by phosphorylation of mTOR on S2448 ^[66][93].

The cyclic adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase A (PKA) is a key regulator of the mTOR pathway, and both are up-regulated in metastatic breast cancer ^[51][78]. PKA consists of two catalytic subunits and two regulatory subunits and can be activated under glucose-deprived conditions where an accumulation of cellular levels of cAMP occurs. Activation of PKA by cAMP occurs when cAMP binds to the regulatory subunits of PKA, which dissociates them from the catalytic subunits that are responsible for PKA activities ^[67][68][69][94,95,96]. Once activated, PKA promotes the phosphorylation and activation of AMPK in an LKB1-dependent manner ^[70][97], which then leads to the inhibition of mTORC1 by phosphorylation of raptor on S791 [98]. The study by Kari ^[71]et al. [97] found that the knockout of LKB1 significantly reduced AMPK phosphorylation in response to PKA activation; however, the reduction in LKB1 did not completely ablate this effect, possibly due to residual LKB1. They also speculated that AMPK phosphorylation is mediated by another kinase. Other than LKB1, Ca²⁺/calmodulin-dependent protein kinase kinase 2 (CaMKK2) ^[72][99] and transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) ^[73][74][100,101] are both known to phosphorylate AMPK on T172.  Since this entry is focused on discussing the role of AMPK in regulating the mTOR pathway, further discussion regarding the regulation of AMPK has been reviewed in detail elsewhere [102].

Clinical trials have assessed the efficacy of rapalogs in combination with trastuzumab for the treatment of HER2-positive breast cancer. In a phase I study, everolimus, trastuzumab, and paclitaxel combination therapy of HER2-positive breast cancer showed promising results with an ORR of 44% and median PFS of 34 weeks (Table 2); however, grade 3 to 4 neutropenia was common in patients ^[113][164]. In the BOLERO-1 phase III clinical trial ^[114][115][165,166], everolimus, trastuzumab, and paclitaxel combination therapy for HER2-positive breast cancer showed an objective response, and median PFS was similar between patients who received the addition of everolimus to the trastuzumab and paclitaxel therapy compared to the addition of the placebo. Interestingly, patients with HER2-positive/hormone receptor-negative (HR−) status showed an improved median PFS compared to the placebo group (20.27 months vs. 13.08 months) (Table 2). In the BOLERO-3 phase III clinical trial [167], everolimus, trastuzumab, and mitotic inhibitor vinorelbine combination therapy of patients with trastuzumab-resistant, HER2-positive breast cancer showed minor improvements in response and median PFS (Table 2). In both BOLERO-1 and BOLERO-3 trials, toxicity was more frequent for patients receiving everolimus than in the placebo groups. Another rapalog, ridaforolimus, used in combination with trastuzumab therapy showed some success in clinical benefit rates and median PFS (Table 2) with modest toxicities [168]. In the LCCC 1025 phase II clinical trial [169], everolimus, trastuzumab, and vinorelbine combined therapy for HER2-positive breast cancer patients with brain metastases showed similar clinical benefit rates relative to other clinical studies using everolimus for HER2-positive breast cancer therapy (Table 2). Together, these clinical trials observed that combining rapalogs and trastuzumab presented a slight improvement in clinical benefits compared to HER2 inhibition alone. However, mTORC1 inhibition resulted in a higher incidence of low-grade and grades 3–4 toxicities. Furthermore, these studies assessed the efficacy of rapalogs that inhibit mTORC1 and not mTORC2 ^[116][117][170,171]. Inhibition of mTORC1 promotes mTORC2 activation and hence mTORC2-dependent feedback activation of the Akt signaling pathway ^[91][117][127,171], thus promoting cancer cell survival and proliferation. Interestingly, in preclinical studies and in vitro, O’Brien et al. demonstrated that combined inhibition of Akt and mTOR prevented the feedback activation induced by trastuzumab-mediated inhibition of mTORC1 and sensitized trastuzumab-resistant cells to trastuzumab ^[118][172].

For the dual PI3K-mTOR inhibitors, clinical trials assessing the efficacy of NVP-BEZ235 therapy for prostate cancer ^[110][159], renal cell carcinoma ^[111][160], and solid tumors ^[112][161] observed high toxicity and no improvement in clinical responses. More recently, the maximum tolerated dose of NVP-BEZ235, as well as formulations and dosage forms, were assessed in patients with solid tumors, including those with HER2-positive breast cancer ^[119][173]. Here, Rodon et al. reported that the adverse effects of NVP-BEZ235 monotherapy, in any formulation, and when used in combination with trastuzumab, were similar. The authors noted that the onset of the adverse effects occurred shortly after dosing and may be caused by low absorption and precipitation of the drug at high doses rather than mechanism-based toxicities. Furthermore, certain toxicities associated with PI3K inhibition (hyperglycemia and rash) and mTOR inhibition (pneumonitis) were not observed ^[119][173]. Despite this, an anti-tumor effect with a favorable safety profile could not be achieved with any NVP-BEZ235 treatments or formulations.

Genetically engineered mouse models of HER2-positive breast cancer overexpress Neu/HER2 (wild-type or mutant Neu) in mammary glands under the Mouse Mammary Tumor Virus (MMTV) promoter. The first Neu/HER mouse model, MMTV-neu mice, was initially described in 1988 by the Leder laboratory ^[120][177]. MMTV-neu mice constitutively express Neu/HER2 and produce rapidly growing, highly metastatic mammary tumors ^[120][177] (reviewed in ^[121][178]). Later, in 2008, MMTV-NIC (neu-IRES-Cre) mice were generated, which simultaneously expresses neu and cre recombinase (activated Neu/HER2-MMTV-Cre) under the endogenous Erbb2 promoter. NIC mice produce aggressive HER2-positive mammary tumors at around 146 days old ^[122][179].

When human breast cancer tissue microarrays were analyzed for expression of LKB1, a critical negative regulator of mTORC1, 31% of HER2-positive breast cancers were deficient in LKB1 expression ^[50][77]. Based on those observations, Lkb1 (also known as Stk11) was knocked out in NIC mice ^[122][179], by genetic crossing of NIC mice with Stk11^fl/fl mice, to generate Stk11^−/−NIC (Lkb1^−/−NIC) mice. The loss of Lkb1 reduced the latency of tumorigenesis in Lkb1^−/−NIC mice compared with NIC mice ^[50][77]. Furthermore, tumors from Lkb1^−/−NIC mice showed enhanced phosphorylation of the S6K1 substrate ribosomal protein (S6), elevated ATP levels, and changes in metabolic enzymes and metabolites indicative of mTORC1 hyperactivation ^[50](Figure 2) [77]. Chen et al. ^[123][180] assessed the role of LKB1 in breast cancer, including HER2-positive breast cancers, using immunohistochemical analysis of tumors from early breast cancer patients and in silico analysis obtained from the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC) dataset. Although LKB1 protein expression did not correlate with HER2 status, high LKB1 expression in HER2-positive breast cancer patients correlated with improved overall survival, consistent with the findings of Andrade-Vieira et al. ^[50][77] that found NIC mice expressing LKB1 had an increased tumor latency compared to Lkb1^−/−NIC mice.

Glycolysis uses glucose to generate two molecules of pyruvate and energy in the form of ATP  ^[125](Figure 3) [182] (reviewed in ^[126][183]). Under aerobic conditions, pyruvate is transported into the mitochondria and converted to citrate and CO₂. Citrate goes on to the tricarboxylic acid (TCA) cycle that facilitates the transport of electrons to the electron transport chain (ETC). Electrons generated by the ETC are used for oxidative phosphorylation (OXPHOS) to generate ~36 ATP molecules per glucose molecule (reviewed in ^[127][184]). When oxygen is limited, pyruvate is metabolized via anaerobic glycolysis, which generates ATP less efficiently but 100X more rapidly compared to OXPHOS ^[128][185]. In anaerobic glycolysis, LDH catalyzes the reduction of pyruvate and regeneration of NAD⁺, where pyruvate and NADH are converted to lactate, NAD⁺, and two ATP molecules (Figure 3).

Warburg et al. ^[129][186] made the landmark observation that tumor cells metabolized high levels of glucose to produce ATP and lactate in the presence of oxygen. The metabolic shift of OXPHOS to aerobic glycolysis in tumor cells is known as the “Warburg effect”. This established the paradigm that cancer cells use glycolysis instead of OXPHOS to produce ATP due to impaired OXPHOS function, potentially due to mitochondrial damage. However, contrary to the hypotheses postulated since the discovery of the Warburg effect, mitochondria in most tumor cells are intact and OXPHOS is not impaired in most cases ^[130][187]. Recent evidence indicates that OXPHOS and aerobic glycolysis do function in cancer cells [188]. Using a population of cancer stem cells (CSC) from ^[131]epithelial ovarian cancer, Pasto et al. [189] found that the expression of many essential enzymes involved in TCA and ETC were up-regulated (e.g., citrate synthase (CS), isocitrate dehydrogenase (IDH2), ATP5B, HKII, PKM, and PFK) concomitant with the up-regulation of GLUT1 cell surface levels in CSCs (CD44⁺CD117⁺) compared to non-tumorigenic CD44⁺CD117⁻ cells. The CSCs showed characteristics consistent with cells that use OXPHOS such as elevated generation of mitochondrial reactive oxygen species (ROS), increased membrane potential, and ETC inhibition, resulting in apoptosis. Through evaluation of metabolites isolated from three types of Kras^G12D-driven non-small cell lung cancer (NSCLC) mouse models and xenograft models using A549 and H1975 lung cancer cell lines, Davidson et al. [188] observed that the metabolism of lung tumors relied on oxidative metabolism and aerobic glycolysis. Tumors from NSCLC mice showed increased glucose uptake concomitant with increased TCA cycle metabolism. Increases in the levels of lactate were observed in lung tumors of two of the three NSCLC models and the tumors of xenograft mice relative to normal tissues. Furthermore, tumor growth rates differed between the lung tumors from the two NSCLC models, although they both showed high levels of lactate, which argues that lactate levels do not necessarily correlate with proliferation. Interestingly, lung tumor cells isolated from one of the NSCLC mouse models relied more on glutamine metabolism to support cell growth, whereas tumor cells analyzed directly from the same NSCLC mouse model did not rely on glutamine metabolism. The observation of metabolic differences between lung cancer cells in vitro and in vivo suggested a role of the tumor microenvironment in influencing cancer cell metabolism. Interestingly, OXPHOS supports tumor growth through the metabolites generated by glycolysis and the ETC. For example, the generation of aspartate, a precursor used to synthesize purine and pyrimidine, can be metabolized by tumor cells to support growth [190,191]. Moreover, several studies found that the loss of OXPHOS by depletion of mitochondrial DNA (mtDNA) resulted in reduced tumor cell proliferation in vitro and tumorigenicity in vivo [192,193,194]. 

Several studies have observed that resistance to targeted therapies in cancer cells is associated with increased glycolytic activity and expression of glycolytic enzymes ^{[132][133][134]}[195,196,197]. The concept of metabolic reprogramming has gained popularity as a means for tumors to adapt to the metabolic requirements for survival (for further details, please refer to reviews ^[135][136][198,199]).

HER2 overexpression in breast cancer cell lines increased glycolysis as indicated by increased glucose uptake and lactate production, and decreased oxygen consumption rates [200]. Zhao et al. [200] ^[137]found that HER2 overexpression in breast cancer cell lines MCF-7 and MDA-MB-435 increased glycolysis, indicated by increased glucose uptake and lactate production, and decreased oxygen consumption rates. Of the HER2-positive breast cancer subtypes, metabolomics data of patient tumors showed that the HER2-E and basal-like subtypes had elevated levels of glycolytic enzymes G-6-P and F-6-P and lactate compared to the luminal A and luminal B subtypes ^[138][201]. Zhang et al. [202] recently identified that the DNA repair protein Transcriptional repressor zinc finger and BTB domain containing 1 (ZBTB1) was dramatically down-regulated in breast tumors of HER2-positive patients, and that ZBTB1 suppressed HER2 expression by binding to the estrogen receptor alpha (ERα) binding site of the HER2 intron. Re-expression of ZBTB1 in HER2-expressing breast cancer cell lines reduced lactate production, glucose uptake, and down-regulated LDH and HK expression, indicating that the elevated expression of HER2 due to the loss of ZBTB1 promotes aerobic glycolysis [195].  Interestingly, Gale et al. ^[139][203] observed a metabolic shift in HER2-positive cell lines that acquired trastuzumab resistance. Here, trastuzumab-resistant, HER2-positive cells showed an up-regulation of numerous genes encoding enzymes involved in OXPHOS. Although the oxygen consumption rate was similar among parental HER2-positive cell lines and trastuzumab-resistance cells, lactate production was not examined. Trastuzumab-resistant cells and patient data showed increased expression of the ATP synthase subunits ATP5J and ATP5B, which were also found to correlate with poor survival. The addition of an ATP synthase inhibitor re-sensitized tumor cells to trastuzumab in xenograft mouse models of HER2-positive breast cancer. In contrast, studies have observed increased glycolysis, via increased glucose uptake and lactate production, in trastuzumab-resistant, HER2-positive breast cancer cells in vitro and in vivo using xenograft mouse models ^[132][140][195,204]. Aberrant activation of Akt, which is activation by HER2, has been observed to increase glucose consumption and lactate production, indicative of aerobic glycolysis in cancer cells ^[141][205]. Using murine-derived leukemic and human glioblastoma cell lines and xenograft mouse models, Elstrom et al. ^[116][170] demonstrated that overexpression and activation of Akt stimulated aerobic glycolysis as indicated by the increase in glucose consumption and lactate production. This effect was negated by treatment with the Akt inhibitor LY294002 in vitro [205,206] and in vivo [205]. The glycolytic activitiets in HER2-positive breast cancer cell lines are reduced in response to trastuzumab treatment. Together, these studies indicate that activation of the HER2-Akt pathway enhances glycolytic activity. Constitutive activation of mTORC1 was also shown to promote glycolysis. ^[142]Loss of NPRL2, a suggesting tnegative regulator of mTORC1, decreased the levels of TCA metabolites and was concomitant with increases in glucose uptake and lactate production [207]. This suggests that mTOR hyperactivation is a critical contributor to the elevated glycolytic activity observed in HER2-positive breast cancer.

As the HER2-E subtype expresses higher levels of HER2 and glycolytic metabolites compared with other HER2-positive breast cancer subtypes, the HER2-E subtype also displayed higher levels of phosphorylated S6K, a substrate of mTORC1 ^[143][208]. Likewise, since the loss of Lkb1 causes mTORC1 hyperactivation, tumors from the Lkb1^−/−NIC mouse model of HER2-positive mammary cancer showed elevated levels of glycolytic metabolites, including lactate, and up-regulated LDH and PDH expression compared with control wild-type (WT) mice ^[50][77]. Inhibition of mTORC1/mTORC2 (Torin-1, AZD8055) or mTORC1 (rapamycin) down-regulated LDH expression and had little to no effect on PDH expression in the primary tumor cells isolated from Lkb1^−/−NIC mice compared to the cells from NIC mice ^[50][107][77,151]. Furthermore, tumors from AZD8055-treated Lkb1^−/−NIC mice showed reduced glycolytic activity and oxygen consumption rates compared with vehicle-treated mice. Characterization of mitochondrial content, size, and cristae density was greater in mammary tumors from Lkb1^−/−NIC mice compared with mammary glands from control WT mice. AZD8055 treatment reduced the mitochondrial content in Lkb1^−/−NIC but not in control WT mice, indicating the role of mTOR activity in mitochondrial biogenesis in HER2-positive cancer ^[107][151]. This study demonstrates that therapies that simultaneously target mTORC1/mTORC2 and glycolytic metabolism in cancer produce the best therapeutic outcome against HER2-positive breast cancer.

Tumor cells can develop a dependency on glycolysis for survival. Glucose analogs cause glucose deprivation, resulting in the suppression of glycolysis as they cannot be metabolized by cells. 2-deoxy-D-glucose (2-DG) is a glucose analog that is taken into the cytosol through glucose transporters (GLUTs), where hexokinase phosphorylates 2-DG to generate 2-DG-P; however, phosphohexose isomerase is not able to metabolize 2-DG-P any further (reviewed in ^[144][209]). Here, downstream glycolysis and production of cellular ATP are inhibited by the accumulation of 2-DG, which is associated with impaired cell cycle progression and enhanced cell death of tumor cells ^[145][210]. Although 2-DG negatively affects cell cycle progression, studies have demonstrated that the inhibition of glycolysis by 2-DG monotherapy is concomitant with the induction of Akt phosphorylation at T308 and S473 ^[146][147][211,212]. This could have negative implications in the efficacy of 2-DG as a cancer treatment as 2-DG-induced Akt activation would oppose the 2-DG-dependent inhibition of cell proliferation and survival.

Considering that the availability of endogenous glucose can mitigate the efficacy of 2-DG, 2-DG may not completely block glycolysis. However, antiproliferative and cell death-promoting effects of 2-DG have been observed in vitro and in vivo in cancer cells ^[148][213]. Treatment using 2-DG induces endoplasmic reticulum stress, leading to autophagy. This results from the accumulation of misfolded proteins in the ER lumen concomitant with ER stress and the unfolded protein response, a mechanism of inhibiting protein translation to relieve ER stress ^[148][213]. Inhibition of autophagy prevented 2-DG-induced autophagy and ER stress but did not reverse the depletion of ATP. Furthermore, treatment using oxamate, which depletes ATP without inducing ER stress, did not induce autophagy ^[148][213], indicating that 2-DG induces autophagy in cancer cells by increasing ER stress and not by ATP depletion.

Suppression of glycolysis in HER2-positive breast cancer has been observed to reduce HER2-driven mammary tumor cell growth in vitro as well as in vivo with mouse models ^[149][214]. In the Lkb1^−/−NIC mouse model of HER2-positive mammary cancer, 2-DG monotherapy reduced tumor burden and growth compared with vehicle-treated mice, but to a lesser extent than AZD8055 monotherapy ^[107][151]. Tumors from 2-DG-treated mice showed reduced glycolysis, oxygen consumption rate, mitochondrial content, and down-regulation of HK, PDH, and LDH expression. The combination of AZD8055 and 2-DG further augmented these effects, indicating that 2-DG sensitized the tumor cells to mTORC1/2 inhibition. In addition, 2-DG treatment did not significantly affect the phosphorylation status of S6, ACC, AMPK, p90RSK, and ERK compared to vehicle-treated mice. Interestingly, 2-DG induced phosphorylation of AMPK (T172) concomitant with reduced mTORC1 activity as observed from reduced phosphorylation of mTOR (S2448) and S6K1 (T389) in various cell types ^[150](Figure 1) [215]. Hurley et al. reported that 2-DG induced the phosphorylation of AMPK and ACC in HeLa cells that were LKB1^+/+ or LKB1^{−/− [151]}[216], indicating that 2-DG induced another kinase responsible for AMPK phosphorylation and activation independent of LKB1. Inhibition of CaMKK2, which also phosphorylates AMPK on T172, using STO-609 prevented 2-DG-induced AMPK phosphorylation ^[151][152][216,217]. Furthermore, since HeLa cells do not express the CaMKKβ isoforms 1 and 2 ^[153][218], shRNA knockdown of the CaMKKβ3 isoform, but not the CaMKKα isoform, prevented AMPK phosphorylation by 2-DG. Since 2-DG did not significantly induce AMPK or ACC phosphorylation in tumors from Lkb1^−/−NIC mice ^[107][151], this may suggest CaMKKβ3 is not active in this model. Together, these studies suggest that, in addition to inhibiting glycolysis directly through inhibition of phosphohexose isomerase activity, 2-DG inhibits mTORC1-mediated promotion of glycolysis.

Studies assessing the efficacy of 2-DG and other metabolic interventions in combination with various anticancer therapies have observed tolerable adverse effects from the addition of 2-DG ^[154][219] (reviewed in ^[155][220]). Preclinical studies determined that high dosages negatively impacted the respiratory frequency and mean arterial blood pressure ^[156][221], and reversible cardiac toxicity has been associated with 2-DG treatment in rats ^[157][222]. Despite the therapeutic potential of 2-DG for cancer treatment, studies have shown little to no effect of 2-DG treatment alone in inhibiting tumor growth in preclinical mice models, including genetic ^[107][151] and xenograft models ^[158][223]. In a clinical trial conducted in patients with a variety of solid tumors, Raez et al. ^[154][219] observed that patients treated with 2-DG presented with reversible hyperglycemia (100%), gastrointestinal bleeding (6%), and cardiac abnormalities (QTc prolongation; 22%).

As previously discussed, HER2 hyperactivity promotes mTOR pathway-dependent up-regulation of glycolytic enzymes, indicating that mTOR is a major contributor to the enhanced glycolytic activation observed in HER2-positive breast cancer. Inhibition of the mTOR pathway can negate the effect of HER2 hyperactivity, enhancing glycolysis in cancer cells, but prolonged inhibition of mTOR pathway activity leads to an up-regulation of ERK activation ^[81][103][111,147]. Consistent with this, our laboratory observed an up-regulation of ERK phosphorylation, concomitant with a decrease in glycolytic activity and tumor growth, was observed in mammary tumors from Lkb1^−/−NIC mice treated with the mTORC1/2 inhibitor AZD8055 ^[107][151]. Furthermore, mammary tumors from AZD8055-treated mice showed up-regulation of AMP, ACC, and p90RSK phosphorylation. Like mTOR inhibition, 2-DG also suppresses glycolysis but activates PI3K ^[146][147][211,212] and AMPK ^[151][152][216,217]. In aour preclinical study, 2-DG reduced tumor growth to a lesser extent than AZD8055. As AZD8055 did not completely block glycolytic activity in mammary tumors of Lkb1^−/−NIC mice, combination treatment was performed by adding 2-DG to the AZD8055 treatment to further inhibit glycolysis in addition to mTORC1/2. This combination treatment further reduced the mammary tumor volume and burden compared to AZD8055 or 2-DG monotherapies, but more importantly, the addition of 2-DG blocked the AZD8055-dependent induction of ERK phosphorylation, as well as the phosphorylation of AMPK, ACC, and p90RSK ^[107][151]. Sun et al. [224] also observed that 2-DG treatment of lung cancer cells resulted inc a time- and dose-depende thent down-regulation of ERK phosphorylation in an LKB1/AMPK-dependent manner. Lkb1^−/−NICAlthough our mouse model lacks LKB1 expression, 2-DG can induce AMPK independently of LKB1, as previously discussed in Section 4.4.  These studies demonstrate the benefit of combining glycolytic inhibition via 2-DG with mTORC1/2 inhibition, in which the addition of 2-DG prevents the mTOR inhibitor-dependent induction of pro-survival MAPK pathway signaling. Furthermore, Cheng et al. showed 2-DG sensitized 5-fluorouracil (5-Fu)-resistant pancreatic cancer cell lines to the cytotoxic effects of 5-Fu [225]. They revealed that 2-DG-mediated activation of PI3K-Akt was required to sensitize the drug-resistant cells to cell growth inhibition and apoptosis induced by 5-Fu. In the study by Ma et al. ^[159][226], 2-DG was also found to overcome chemo-resistance due, in part, to the suppression of ATP-dependent drug efflux transporters. These studies strongly suggest that the addition of 2-DG may improve the efficacy of therapies using mTOR inhibition, as well as other targeted therapies, by migrating factors that contribute to drug resistance such as activation of pro-survival signaling and drug efflux.