Supplementary Table 1 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

Supplementary Figure Legends 1-3, Methods, Tables 1-4 from Alterations of the HBP1 Transcriptional Repressor Are Associated with Invasive Breast Cancer

ABSTRACT Triple negative breast cancers (TNBC) pose exceptional challenges with fatal brain metastases as a clear and unmet need. Immune checkpoint inhibitors (ICIs) are promising therapeutic strategies, but most TNBC are resistant, or “cold” tumors, due to lack of tumor-resident immune engagement. No FDA-approved therapies exist which promote a “cold-to-hot” transition or induce the important biomarker PD-L1, often used for ICI clinical decision-making. Maximal ICI susceptibility, or a full “cold-to-hot” transition, requires reciprocal Wnt signaling inhibition and Jak/STAT/interferon signaling activation. We report a new compound combination (CHA1) that fits the above criteria. CHA1 is comprised of EGCG (epigallocatechin-3-gallate; green-tea compound) and decitabine (DNA-methyltransferase (DNMT1) inhibitor; 5’deaza-cytidine; FDA-approved for hematologic malignancies). We used immune-compromised and syngeneic TNBC pre-clinical models to investigate tumor-intrinsic and tumor-resident T-cell effects, respectively. All results required CHA1 (but not EGCG or decitabine alone) and utilized attainable human dose equivalences with manageable safety profiles. CHA1 triggered efficient Wnt signaling inhibition by elevating Wnt pathway inhibitors (HBP1 and SFRP1) and traversed the blood-brain barrier to reduce both tumor and brain metastatic growth. Transcriptomic and expression analyses revealed that CHA1 treatment effectuated a robust tumor-intrinsic JAK/STAT/IFN response 1) to induce PDL1 and 2) to induce antigen presentation and processing genes, including MHC-1, MHC-2 and numerous genes attributed to professional antigen-presenting cells; 3) to induce CD8+-T-cell infiltration and activation. Additionally, CHA1 pre-treatment improved anti-PDL1 efficacy in a syngeneic setting. Lastly, we derived a composite gene signature emblematic of CHA1 treatment and of a favorable clinical prognosis in-silico. Together, our work supports a model in which CHA1 influences epigenetics, Wnt and Jak/STAT/IFN signaling mechanisms—all to reprogram an epithelial-mesenchymal TNBC tumor to express antigen-presenting properties and to recruit and activate tumor-resident CD8 + -T-cells. We discuss our findings in the context of cancer biology and immunity with implications for improving ICI susceptibility for TNBC.

Epilepsy is a complex neurological condition characterized by repeated spontaneous seizures and can be induced by initiating seizures known as status epilepticus (SE). Elaborating the critical molecular mechanisms following SE are central to understanding the establishment of chronic seizures. Here, we identify a transient program of molecular and metabolic signaling in the early epileptogenic period, centered on day five following SE in the pre-clinical kainate or pilocarpine models of temporal lobe epilepsy. Our work now elaborates a new molecular mechanism centered around Wnt signaling and a growing network comprised of metabolic reprogramming and mTOR activation. Biochemical, metabolomic, confocal microscopy and mouse genetics experiments all demonstrate coordinated activation of Wnt signaling, predominantly in neurons, and the ensuing induction of an overall aerobic glycolysis (Warburg-like phenomenon) and an altered TCA cycle in early epileptogenesis. A centerpiece of the mechanism is the regulation of pyruvate dehydrogenase (PDH) through its kinase and Wnt target genes PDK4. Intriguingly, PDH is a central gene in certain genetic epilepsies, underscoring the relevance of our elaborated mechanisms. While sharing some features with cancers, the Warburg-like metabolism in early epileptogenesis is uniquely split between neurons and astrocytes to achieve an overall novel metabolic reprogramming. This split Warburg metabolic reprogramming triggers an inhibition of AMPK and subsequent activation of mTOR, which is a signature event of epileptogenesis. Interrogation of the mechanism with the metabolic inhibitor 2-deoxyglucose surprisingly demonstrated that Wnt signaling and the resulting metabolic reprogramming lies upstream of mTOR activation in epileptogenesis. To augment the pre-clinical pilocarpine and kainate models, aspects of the proposed mechanisms were also investigated and correlated in a genetic model of constitutive Wnt signaling (deletion of the transcriptional repressor and Wnt pathway inhibitor HBP1). The results from the HBP1 -/- mice provide a genetic evidence that Wnt signaling may set the threshold of acquired seizure susceptibility with a similar molecular framework. Using biochemistry and genetics, this paper outlines a new molecular framework of early epileptogenesis and advances a potential molecular platform for refining therapeutic strategies in attenuating recurrent seizures.

Abstract Excessive Wnt signaling is associated with 1) poor prognosis in triple-negative breast cancer (TNBC) and other cancers and 2) immune checkpoint inhibitor resistance, thus limiting its therapeutic application. From our previous work and Wnt signaling principles, we devised CHA1 as an inhibitor of Wnt signaling. CHA1 combines the green tea catechin EGCG (Epigallocatechin-3-gallate) and the DNA methyltransferase inhibitor decitabine, which have been used in numerous clinical trials and/or FDA approved for other cancers, respectively. Our investigations showed that CHA1 treatment (but not EGCG or decitabine alone) reduced primary tumors and metastases in TNBC xenograft and decreased Wnt signaling. Unexpectedly, CHA1 reprogrammed intrinsic tumor properties for antigen presentation and immune cell infiltration. The implications governing tumor-immune cell interactions are discussed in the context of increasing checkpoint inhibitor susceptibility. Both Immune-compromised and immune-competent TNBC preclinical models were used for mechanistic elaboration. In CHA1-treated human TNBC tumors in immune-compromised mice, biochemical and RNA seq analyses show that Wnt signaling was decreased due to induction of Wnt pathway inhibitors (e.g., SFRP1, DKK1, HBP1). The RNA seq analysis also revealed induction of >100 genes for antigen presentation and associated processes. MHC staining of CHA1-treated tumors verified the genotypic changes, which were also accompanied by robust gamma interferon signaling. All results were recapitulated in the immune-competent 4T1 syngeneic TNBC model. We observed large increases in tumor-infiltrating CD8+ T cells with CHA1 treatment. Lastly, recent reports collated the molecularly disparate properties of a “cold-to-hot” transition, in which “hot” tumors have increased immune cell infiltration and sensitivity to checkpoint inhibitors. Remarkably, the TNBC tumors (typically “cold”) that were treated with CHA1 now exhibited the unrelated list of “hot” tumor properties: 1) epigenetic reprogramming, 2) suppressed Wnt signaling, 3) E-cadherin and epithelial marker re-expression, 4) CD8+ T-cell enrichment, 5) increased tumor antigen presentation properties, and 6) increased tumor PD-L1 expression. The CHA1 mechanism is consistent with a global reprogramming of intrinsic tumor properties, triggered by differential Wnt and interferon signaling. CHA1 engages the fundamental processes that regulate tumor-immune cell dynamics, which, in turn, govern immune cell infiltration and determine checkpoint inhibitor sensitivity. Our work additionally establishes a molecular framework for assessing compounds that engage a fundamental “cold-to-hot” tumor reprogramming and that may predict new immune checkpoint inhibitor sensitivity. Thus, CHA1 treatment may reprogram tumor-immune cell dynamics to significantly expand the spectrum of TNBC and other tumors that can be efficaciously treated with immune checkpoint inhibitors (e.g., anti-PDL1 and/or anti-PD1). Citation Format: Mariam Alamoudi, Mollie Chipman, Francesca Deleso-Frechette, Eileen Liu, Rui Zhang, Zixu Wang, K. Eric Paulsopn, Amy S. Yee. A therapeutic strategy to inhibit Wnt signaling also reprograms breast tumor-immune cell interactions: Perspectives for conferring immune checkpoint inhibitor susceptibility [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A39.

Infantile spasms (IS) are a catastrophic childhood epilepsy syndrome characterized by flexion-extension spasms during infancy that progress to chronic seizures and cognitive deficits in later life. The molecular causes of IS are poorly defined. Genetic screens of individuals with IS have identified multiple risk genes, several of which are predicted to alter β-catenin pathways. However, evidence linking malfunction of β-catenin pathways and IS is lacking. Here, we show that conditional deletion in mice of the adenomatous polyposis coli gene (APC cKO), the major negative regulator of β-catenin, leads to excessive β-catenin levels and multiple salient features of human IS. Compared with wild-type littermates, neonatal APC cKO mice exhibit flexion-extension motor spasms and abnormal high-amplitude electroencephalographic discharges. Additionally, the frequency of excitatory postsynaptic currents is increased in layer V pyramidal cells, the major output neurons of the cerebral cortex. At adult ages, APC cKOs display spontaneous electroclinical seizures. These data provide the first evidence that malfunctions of APC/β-catenin pathways cause pathophysiological changes consistent with IS. Our findings demonstrate that the APC cKO is a new genetic model of IS, provide novel insights into molecular and functional alterations that can lead to IS, and suggest novel targets for therapeutic intervention.

: Epileptogenesis is a gradual process by which normal brain transforms into one that sustains seizures. It is instigated by an inciting event (e.g. prolonged seizure called status epilepticus (SE), head injury, infection or stroke). This is followed by a variable (months to years in humans) latent period followed by the emergence of spontaneous seizures, with potential for later mood and learning disabilities. While the latent period is a time during which behavioral seizures are not observed, it is a period of tissue and cellular remodeling that sets up the development of chronic seizure activity, or epilepsy. In this grant, we have drawn expertise from other fields to discover new mechanistic insights into epileptogenesis. In the past year, we have expanded our understanding of molecular mechanisms and uncovered new possible insights for therapeutics with a drug combination that we had been developing for breast cancer treatment. Surprisingly, the combination attenuates seizures in two different models of temporal lobe epilepsy.

: Triple negative breast cancer (TNBC) represents 20-25% of sporadic breast cancers, lacks ER, PR, and overexpressed Her2 and thus has no targeted treatment options. TNBC is the most clinically challenging subtype with exceptionally poor prognosis, high recurrence and metastases and currently represents an unmet medical need. While classified pathologically by negative criteria, TNBC is a heterogeneous group of breast cancers in need of better molecular classification. Numerous studies have linked EGFR signaling to TNBC, but paradoxically, TNBC is refractory to the well-used EGFR inhibitors that have been efficacious in other cancers. In addition, Wnt signaling has been linked to TNBC, but its relationship to EGFR signaling and inhibitor resistance has not been explored. In the current work, the HBP1 transcriptional repressor may be a new and unappreciated bridge to both TNBC pathways. Previously, we have shown that decreases in HBP1: 1) trigger an increase in both Wnt and EGFR signaling to regulate proliferation and senescence; and 2) were coincident with exceptional increased tumor growth and invasiveness in preclinical models of breast cancer. Our new data show that HBP1 levels are significantly reduced in TNBC relative to other breast cancer subtypes in clinical specimens. Strikingly, a new combined EGFR and HBP1 gene signature predicted 90+% of TNBC patients in 4 large patient databases and predicted a poor patient prognosis in non-TNBC patients. To understand how HBP1 decreases trigger a poor prognosis, a whole genome analysis revealed a surprising change in several hundred genes to enact a Warburglike metabolic reprogramming. The Warburg effect is characterized by increased glycolytic flux with increased biosynthesis of amino acids, lipids, and nucleic acids--all to provide for the increased growth and proliferation demands of a tumor cell. This grant seeks to understand how HBP1, EGFR and Wnt signaling trigger metabolic reprogramming in the context of TNBC.

s: American Epilepsy Society Meeting 2011, 2012, 2013. Funding applied: DOD grants, CURE foundation grants (not funded). Successful funding of CURE foundation dream team grant on Infantile Spasms: Some preliminary data went into a successful multi-investigator CURE foundation grant on infantile spasms. Our discovery of Wnt signaling increases in epileptogenesis set the stage for the new research project. The team is Prof. Chris Dulla, Amy Yee, and Chris Dulla (PI). Dr. Audrey Yee had a key early role. Dr. Audrey Yee correctly identified Aicard’s like syndromes in a cAPCKO mice, predicted to have elevated Wnt signaling (owned by Dr. Michele Jacob). Drs. Amy Yee, Dulla and Jacob worked as a team to develop the project and investigate APC and Wnt signaling as an etiology for IS. Dr. Amy Yee created an elegant Infantile and Wnt signaling framework on which to evaluate and develop the multidisciplinary results. The working hypothesis incorporated the genetics of IS and numerous concepts of Wnt signaling gleaned from the cancer and developmental biology field. Remarkably, Dr. Yee noticed that several genes genetically linked to IS were directly linked to different aspects of Wnt pathway function, leading us to hypothesize that interference with Wnt signaling functions may be an excellent therapeutic strategy. Dr. Chris Dulla is analyzing the in vitro electrophysiology and in vivo EEG underlying the development of infantile spasms in the cAPCKO mice. The green tea/Decitabine regimen (described here) and other drugs that interfere with Wnt signaling will also be tested for efficacy on IS. CONCLUSION. These studies establish Wnt signaling and its metabolic network as a new set of molecular and therapeutic targets for the etiology of SE and potentially for epilepsy. The scientific discoveries underscore the importance of this expanded network and begin to advance the notion that the early period of epileptogenesis may recapitulate aspect s of brain development. The recapitulation to a period in earlier development has been a hallmark of diseases such as cancer. Our data would support models in the field in which epilepsy is a result of excessive stem cell proliferation and then abnormal differentiation—which together, set up a pathological environment that sustains seizures. Our observations that excessive glucose usage may contribute suggests that novel interventions such as the ketogenic diet may attenuating effects, Our studies also define a novel regiment of drugs in clinical uses that block Wnt signaling attenuates SE induced by two distinct means. These observations underscore the generality of Wnt signaling and provide proof-of-principle that intervening in Wnt signaling may be efficacious for modifying the course of epileptogenesis to prevent recurrent seizures. The Wnt pathway is under intense therapeutic development for cancer and other diseases. By defining the pre-clinical frameworks for epilepsy, this provides an ideal future opportunity to test drugs that attenuate Wnt signaling for their efficacy in disease modification for epilepsy.

Abstract Triple-negative breast cancer (TNBC) represents 20-25% of sporadic breast cancers, lacks ER, PR, and overexpressed Her2 – and thus has no targeted treatment options. TNBC is the most clinically challenging subtype with exceptionally poor prognosis, high recurrence and metastases and currently represents an unmet medical need. While classified pathologically by negative criteria, TNBC is a heterogeneous group of breast cancers in need of improved molecular classification. Numerous studies have linked EGFR signaling to TNBC, but paradoxically, TNBC is refractory to the well-used EGFR inhibitors that have been efficacious in other cancers. In addition, Wnt signaling has been linked to TNBC, but its relationship to EGFR signaling and inhibitor resistance has not been explored. In the current work, the HBP1 transcriptional repressor may be a new and unappreciated bridge to both TNBC pathways. Previously, we have shown that decreases in HBP1: 1) triggered an increase in both Wnt and EGFR signaling to regulate proliferation and senescence; 2) were coincident with exceptional increased tumor growth and invasiveness in preclinical models of breast cancer; and 3) were associated with a poor breast cancer prognosis. While altered metabolism has been investigated in many cancers, the mechanisms by which EGFR and Wnt signaling might trigger a metabolic reprogramming have not been investigated in TNBC. Our data suggests that HBP1 may be a new integrating factor. Our new data show that HBP1 levels are significantly reduced in TNBC relative to other breast cancer subtypes in clinical specimens. We derived a new combined EGFR and HBP1 gene signature that predicted 90+% of TNBC patients in 4 large patient databases and additionally predicted a poor patient prognosis in non-TNBC patients. To understand how HBP1 decreases trigger a poor prognosis, a whole genome analysis revealed a surprising change in 300+ genes to enact a Warburg-like metabolic reprogramming. The Warburg effect is characterized by increased glycolytic flux with increased biosynthesis of amino acids, lipids, and nucleic acids'all to provide for the increased growth and proliferation demands of a tumor cell. The specific pattern was an increase in certain isoforms involved in glycolysis and TCA cycle and a decrease in the expression of genes involved in oxidative phosphorylation. We used gene-, biochemistry- and NMR-based approaches to investigate the metabolic alterations upon changes in HBP1 expression in TNBC cells and tumors. Our results show that hexokinase 2 (HK2), Pyruvate Kinase-M2 (PKM2), Lactate Dehydrogenase A (LDHA), malate dehydrogenase 2 (MDH2) are amongst the metabolic enzymes that are altered with HBP1 expression and with increased Wnt signaling. HK2, PKM2, LDHA and MDH2 have all been previously associated with the Warburg effect. The NMR and biochemical analysis showed that lactate and fumarate are amongst the metabolites that are altered. The results here provide important metabolomics information to impact MRI/MRS studies. While breast MRIs are used for assessing tumor morphology, MRS (magnetic resonance spectroscopy) methods can non-invasively visualize metabolic spectra in tumors or metastases. The metabolic changes from our studies potentially focus MRS/MRI applications for the future discovery of non-invasive MRS biomarkers to improve TNBC classification and treatment efficacy. (Supported by a grant to ASY and JB from the Dept. of Defense). Citation Format: Amy S. Yee, Kurtz Eric Paulson, Kai Wang, Wesley Field, Brian Pedro, Davis Vigneault, James B. Baleja. The HBP1 transcriptional repressor: An unexpected window on breast cancer metabolism in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr B030.

Background and Goals: Triple negative breast cancer (TNBC) represents 15-20% of sporadic breast cancers, lacks ER, PR, and HER2 overexpression - and thus has no targeted treatment options. TNBC is a clinically challenging subtype with exceptionally poor prognosis, high recurrence and metastases and currently represents an unmet medical need. A critical aspect to improving the outcome for TNBC and other breast cancer patients is the prevention and/or treatment of distant metastases, especially to brain. Another major limitation of TNBC treatment is that most current drugs do not cross the blood-brain barrier to target fatal brain metastases, so novel therapeutic approaches are urgently needed. Results: TNBC have a Wnt signaling etiology and our recent work shows that HBP1 (a transcriptional repressor that suppresses Wnt signaling) was decreased in TNBC. Specifically, we showed that the HBP1 gene was mutated or reduced in invasive breast cancers. The under-expression of two Wnt pathways inhibitors (SFRP1 and HBP1) predicted an exceptionally poor prognosis. We sought to identify new therapeutic strategies aimed at TNBC and at decreasing Wnt signaling. Here, we investigated a combined regimen of the green tea polyphenol epigallocatechin gallate (EGCG) and of decitabine (DAC) that we reasoned might be effective at elevating SFRP1 and HBP1 to reverse a poor prognosis. We reported that EGCG decreased Wnt signaling in vitro by elevating HBP1. EGCG is in numerous clinical trials for breast and other cancers. Decitabine is an inhibitor of DNA methylation and is FDA-approved for hematologic malignancies. Thus, new pre-clinical studies have excellent potential to re-purpose existing drugs for TNBC. The combined regimen of EGCG/DAC significantly reduced TNBC tumorigenesis in two orthotopic TNBC tumor xenograft models (MDA-MB231 and Sum149). The inhibition by EGCG/DAC treatment correlated with reduced Wnt signaling and with the elevated SFRP1 and HBP1 gene expression. Importantly, a stable knockdown of either HBP1 or SFRP1 resulted in larger TNBC tumors that were now refractory to EGCG/DAC treatment. These data suggests that the decreased Wnt signaling and elevation of HBP1 or SFRP1 were critical factors to the efficacy of EGCG/DAC. Finally, EGCG/DAC treatment was effective at reducing brain metastases initiated from the mammary orthotopic site in a variant MDA-MB231TNBC tumor model. Conclusions: Our studies indicated that a combined regimen of EGCG and DAC decreased Wnt signaling and reduced TNBC tumorigenesis and resulting brain metastases in pre-clinical models. Thus, EGCG/DAC appears to uniquely cross the blood-brain barrier to reduce the otherwise fatal brain metastases, likely by reducing Wnt signalling. Together, EGCG/DAC may be an excellent therapeutic combination for clinical trials aimed at improving TNBC patient outcomes. (Supported by NIH, AICR, the Komen Foundation, and DOD to ASY, GES, and JB. MC was supported by NIH Hematology-Oncology Training Grant to Tufts Medical Center). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B260. Citation Format: Amy S. Yee, Kurtz Eric Paulson, Maricel Castaner, Helen Uong, Nora Minerva, Kai Wang, Kelsey Gilchrist, Maria Choi, Gail E. Sonenshein, Jim Baleja. Wnt signaling, triple-negative breast cancer, and green tea. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B260.

Inflammatory Breast Cancer (IBC) is a highly aggressive form of cancer characterized by high rates of proliferation, lymphangiogenesis and metastasis, and an overall poor survival. As regular green tea consumption has been associated with improved prognosis of breast cancer patients, including decreased risk of recurrence, here the effects of the green tea polyphenol epigallocatechin-3-gallate (EGCG) were tested on two IBC lines: SUM-149 and SUM-190. EGCG decreased expression of genes that promote proliferation, migration, invasion, and survival. Consistently, growth, invasive properties, and survival of IBC cells were reduced by EGCG treatment. EGCG also reduced lymphangiogenesis-promoting genes, in particular VEGF-D. Conditioned media from EGCG-treated IBC cells displayed decreased VEGF-D secretion and reduced ability to promote lymphangiogenesis in vitro as measured by hTERT-HDLEC lymphatic endothelial cell migration and tube formation. Tumorsphere formation by SUM-149 cells was robustly inhibited by EGCG, suggesting effects on self-renewal ability. Stem-like SUM-149 cells with high aldehyde dehydrogenase (ALDH) activity, previously implicated in poor patient prognosis, were isolated. EGCG treatment reduced growth and induced apoptosis of the stem-like SUM-149 cells in culture. In an orthotopic mouse model, EGCG decreased growth of pre-existing tumors derived from ALDH-positive stem-like SUM-149 cells and their expression of VEGF-D, which correlated with a significant decrease in peritumoral lymphatic vessel density. Thus, EGCG inhibits the overall aggressive IBC phenotype. Reduction of the stem-like cell compartment by EGCG may explain the decreased risk of breast cancer recurrence among green tea drinkers. Recent clinical trials demonstrate the efficacy of green tea polyphenol extracts in treatment of prostate cancer and lymphocytic leukemia with low toxicity. Given the poor prognosis of IBC patients, our findings suggest further exploration of EGCG or green tea in combinatorial treatments against active IBC disease or in maintenance regimens to avoid recurrence is warranted.

The activity of DNA methyltransferase 1 (DNMT1) is associated with diverse biological activities, including cell proliferation, senescence, and cancer development. In this study, we demonstrated that the HMG box-containing protein 1 (HBP1) transcription factor is a new repressor of DNMT1 in a complex mechanism during senescence. The DNMT1 gene contains an HBP1-binding site at bp -115 to -134 from the transcriptional start site. HBP1 repressed the endogenous DNMT1 gene through sequence-specific binding, resulting in both gene-specific (e.g., p16(INK4)) and global DNA hypomethylation changes. The HBP1-mediated repression by DNMT1 contributed to replicative and premature senescence, the latter of which could be induced by Ras and HBP1 itself. A detailed investigation unexpectedly revealed that HBP1 has dual and complex transcriptional functions, both of which contribute to premature senescence. HBP1 both repressed the DNMT1 gene and activated the p16 gene in premature senescence. The opposite transcriptional functions proceeded through different DNA sequences and differential protein acetylation. While intricate, the reciprocal partnership between HBP1 and DNMT1 has exceptional importance, since its abrogation compromises senescence and promotes tumorigenesis. Together, our results suggest that the HBP1 transcription factor orchestrates a complex regulation of key genes during cellular senescence, with an impact on overall DNA methylation state.

Objectives: Overexpression of the epidermal growth factor (EGF) receptor (EGFR) gene in the squamous cell carcinomas of the head and neck (SCCHN) is often associated with inauspicious prognosis and poor survival. N-acetylcysteine (NAC), a compound from some vegetables and allium species, appears anti-tumorigenesis, but the underlying mechanism is unclear. The objective of this study is to investigate the role of NAC in EGFR-overexpressing oral cancer. Materials and methods: Both HSC-3 and SCC-4 human tongue squamous carcinoma cell lines and an HSC-3 xenograft mouse model were used to test the anti-growth efficacy of NAC in vitro and in vivo, respectively. Results: NAC treatment suppressed cell growth, with concomitantly increased expression of HMG box-containing protein 1 (HBP1), a transcription suppressor, and decreased EGFR/Akt activation, in EGFR-overexpressing HSC-3 oral cancer cells. HBP1 knockdown attenuated the growth arrest and apoptosis induced by NAC. Lastly, NAC and AG1478, an EGFR inhibitor, additively suppressed colony formation in HSC-3 cells. Conclusion: Taken together, our data indicate that NAC exerts its growth-inhibitory function through modulating EGFR/Akt signaling and HBP1 expression in EGFR-overexpressing oral cancer.