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Metabolic reprogramming and metabolic plasticity allow cancer cells to fine-tune their metabolism to adapt to the ever-changing environments of the metastatic cascade, for which lipid metabolism and oxidative stress are of particular importance. Continuous production of NADPH, a cornerstone of both lipid and redox homeostasis, is essential for proliferation and cell survival suggesting that cancer cells may require larger pools of NADPH to efficiently metastasize. NADPH is recycled through reduction of NADP+ by several enzymatic systems in cells; however, de novo NADP+ is synthesized only through one known enzymatic reaction, catalysed by NAD+ kinase (NADK). Here, we show that NADK is upregulated in metastatic breast cancer cells enabling de novo production of NADP(H) and the expansion of the NADP(H) pools thereby increasing the ability of these cells to adapt to the oxidative challenges of the metastatic cascade and efficiently metastasize. Mechanistically, we found that metastatic signals lead to a histone H3.3 variant-mediated epigenetic regulation of the NADK promoter, resulting in increased NADK levels in cells with metastatic ability. Together, our work presents a previously uncharacterized role for NADK as an important contributor to breast cancer progression and suggests that NADK constitutes an important and much needed therapeutic target for metastatic breast cancers.

Ovarian cancer accounts for the fifth most common cause of cancer death among women, despite being relatively rare. This is in large because majority of cases are diagnosed at advanced stage and frequently recur after primary treatment. Despite numerous efforts to develop effective targeted therapies and immunotherapies for ovarian cancer the standard of care remains chemotherapies. We postulate that part of the problem is the paucity of preclinical research and clinical trials focused on dormant persister ovarian cancer cells that remain after chemotherapy treatments. Emerging evidence supports the idea that persister cancer cells undergo a period of dormancy following chemotherapy which enables their survival and drive late recurrence. Thus, there is dire need to understand the pathways that empower the establishment of dormancy following chemotherapies in ovarian cancer. Epigenetic reprogramming is notoriously known to enable cell fate transitions and the establishment of drug resistance. An important layer of epigenetic regulation frequently overlooked in cancer is the regulation of nucleosome composition via histone variants. Importantly, analysis of data from the human protein atlas revealed that histone H3.3 chaperone, Histone Regulator Protein A (HIRA) expression inversely correlated with poor prognosis, suggesting that HIRA suppression might play a role in the development of chemotherapy resistance. Here, we demonstrate that HIRA is a major regulator of cell fate transitions in ovarian cancer cells in response to chemotherapies. Our data indicates that HIRA levels are suppressed in response chemotherapies in cancer cells. Genetic suppression of HIRA in ovarian cancer cells triggered a non-poliferative state characterized by the induction of canonical dormancy markers (p27, an increase in p38 phosphorylation) and cell cycle arrest. Accordingly, HIRA suppression in these cancer cells renders them less sensitive to the standard of care chemotherapy agent paclitaxel. Together, our data demonstrates that HIRA levels play an important role in controlling cell fate decisions in ovarian cancer and regulate chemotherapy sensitivity. Citation Format: Maya Yasukawa, Stanislav Drapela, Didem Ilter, Mian M. Shahzad, Ana P. Gomes. HIRA suppression is a mechanism of chemotherapy resistance in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB281.

Changes in the number of copies of the genome, i.e. changes in ploidy are widespread among cancer cells. Increased ploidy is thought to benefit cancer cells by conferring robustness against deleterious consequences of genomic instability. However, whether there are any potential costs associated with increased ploidy has not been sufficiently investigated. One hypothesis holds that high ploidy cells may exhibit increased sensitivity to limitations in resources, due to the extra energy required for the replication of a larger genome. To address this hypothesis, we developed an integrated in vitro and in silico system to investigate the response to glucose restriction in diploid and tetraploid cells. Near diploid (2N) SUM159 breast cancer cells were fused to generate an isogenic tetraploid (4N) cell line. 2N or 4N cells were plated with varying initial concentrations of glucose. Cells grew for five days and media was not refreshed, so cells depleted the glucose in the media until they eventually died. Throughout the experiments, glucose concentration was measured at 24 hour intervals, and images were taken at 8 hour intervals allowing quantification of the number of alive or dead cells over time. These in vitro data were then used as inputs to the in silico portion of our system, which consists of an Ordinary Differential Equation (ODE) model of cancer cell growth. ODE model parameters were estimated using the measured outputs from the in vitro experiments. Parameters estimated from the ODE model gave insight into differences between the 2N and 4N cell lines. We found that 4N cells outcompeted 2N cells at high glucose concentrations, due to their ability to divide more rapidly when glucose was abundant. However, 2N cells were able to outcompete 4N cells at lower glucose concentrations. The advantage of 2N cells at low glucose was not due to an increased proliferative capacity. Instead, 2N cells were more resistant to cell death when glucose was limited. Our next steps will be to expand our system to assess ploidy specific sensitivity to other resources, such as phosphate or oxygen. The ODE model we have developed allows us to identify resource conditions that will select for tumor cells based on ploidy. In future this knowledge can be used to suggest therapeutics that can modulate resource availability within tumors, in order to steer tumor evolution towards less aggressive phenotypes. Citation Format: Richard J. Beck, Didem Ilter, Ana P. Gomes, Noemi Andor. Detecting ploidy specific vulnerability to resource restriction: An integrated in vitro and in silico approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3171.

Lung cancer is the second most diagnosed type of cancer and the leading cause of cancer-related mortality. 85% of all lung cancer cases are non-small cell lung cancer (NSCLC), a large proportion of which are only diagnosed at advanced stages (II-IV) due to lack of early diagnostic techniques. Standard first-line treatment for late-stage inoperable NSCLC still is chemotherapy regiments. However, most NSCLC patients fail to respond to these regimens or relapse upon treatment. Thus, there is a pressing need to identify novel therapeutic targets that can more effectively treat NSCLC. Quinolinic acid phosphoribosyl transferase (QPRT) is a rate-limiting enzyme in the tryptophan catabolic pathway which fuels de novo NAD+ production, whose expression as previously been shown to have a prognostic value in certain cancers. Here, we demonstrate that high QPRT levels is a feature of a large proportion of NSCLC cell lines and that QPRT induction occurs in genetically engineered models of NSCLC (KP and KL) when compared to the normal lung. Considering the critical role of NAD+ levels to enable high rates of proliferation, we hypothesized that QPRT induction enables lung cancer cells to thrive. Strikingly, QPRT knockdown in a panoply of NSCLC cell lines results in pronounced suppression of tumor cells both in 2D and 3D conditions. Interestingly, we observe that QPRT suppression does not affect proliferation or cell cycle progression but rather due to pronounced induction of cell death. Surprisingly, while we observed DNA damage upon QPRT suppression, NAD+ levels were not affected by QPRT suppression indicating that QPRT’s effects in NSCLC are independent of its contribution to the NAD+ pools. Together, our results point for QPRT as a novel and effective therapeutic target for NSCLC. Citation Format: Hossein Kashfi, Nathan Ward, Didem Ilter, Stanislav Drapela, Aimee Falzone, Stephen Gardell, Gina M. DeNicola, Ana P. Gomes. Quinolinic acid phosphoribosyl transferase (QPRT) is an essential liability of non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2161.

Most cancer-related deaths are caused by recurrent metastatic disease with latency periods that range from years to even decades. This halt in disease progression is driven by cancer dormancy, a stage in which residual disease is present but remains asymptomatic before reawakening and often causes lethal forms of the disease. Cell fate decisions, such as the ones that underlie dormancy and reawakening of cancer cells, often require extensive epigenetic remodeling, which in turn allows for the expression of specific genetic programs that trigger cell fate decisions. Therefore, epigenetic remodeling is likely essential for the switch between dormant and proliferative states throughout the metastatic process. Here we focus on epigenetic regulator, histone H3.3 chaperone HIRA and its role in the proliferation-to-dormancy switch in breast cancer cells. In this study, we demonstrate that HIRA suppression leads to cell cycle arrest and induces canonical dormancy pathways via phosphorylation of p38 and induction of p27 expression. In addition, HIRA suppression triggers induction of stress-inducible transcription factors NRF2, MITF and ATF3 to enable the activation of genetic programs that maintain homeostasis and promote survival in dormant cells. Our data also portray that HIRA is suppressed in the well-established isogenic models of dormancy when compared to their metastatic counterparts. Altogether we present for the first-time histone chaperone HIRA as a major regulatory point of cell fate for disseminated cancer cells by controlling their transition between dormant and proliferative states, and therefore suggest HIRA as a novel and tremendously needed therapeutic target for metastatic breast cancer. Citation Format: Stanislav Drapela, Devesh Raizada, Ananya Mukherjee, Joanne Tejero, Didem Ilter, Jose Javier Bravo-Cordero, Ana P Gomes. HIRA is a master regulator of the proliferation-to-dormancy switch in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 65.

Lung cancer is the most common cause of cancer death worldwide, with a five-year survival rate of only 18.6%. Aging is a well-established risk factor for lung cancer development. However, our understanding of how the aging process affects lung cancer remains cripplingly small. In this study, we aim to bridge this gap by evaluating the effects of glucocorticoid hormones (e.g. cortisol), which become chronically upregulated during the aging process, in lung cancer cells. Phenotypically, our studies revealed that chronic exposure to age-related levels of cortisol trigger a mesenchymal-like senescent state in lung cancer cells that favors motility and invasiveness. Acquisition of pro-aggressive features are generally recognized to be driven by epigenetic regulation. Epigenetic regulation occurs at several levels, but perhaps one of the most fundamental levels is nucleosome remodeling via histone variants and their cognate chaperones. Histone H3 variants are critical regulators of nucleosome stability and have a highly specialized histone chaperone network when compared to other histones. Strikingly, we observed that chronic exposure of lung cancer cells to cortisol leads to a pronounced decline in canonical histone levels, which was accompanied by a 40% increase in chromatin accessibility genome-wide. Chromatin accessibility by transcriptional machinery is heavily influenced by histone variants, thus, we hypothesized that cortisol also regulates deposition of histone H3 variants into chromatin. The CAF-1 complex deposits the canonical H3 variants (H3.1/H3.2) onto DNA in a cell-cycle-dependent manner while other histone chaperones, HIRA at genic and DAXX at telomeric regions, deposit the H3.3 variant independently of cell cycle. In accordance with the decline in canonical histone levels, treatment with cortisol suppressed the expression of the CAF-1 complex. Reduced canonical histone abundance is known to trigger “gap filling” of naked DNA with the H3.3 variant to maintain integrity. In support of the idea that H3.3 becomes enriched in chromatin upon chronic exposure of lung cancer cells to cortisol, our results showed that the proportion of H3.3 increases upon cortisol exposure, and concomitantly H3.3 chaperone HIRA is induced upon treatment with cortisol. These data support a model in which chronic exposure to glucocorticoids, as it occurs with aging or through treatment with synthetic glucocorticoids (e.g. dexamethasone), regulates nucleosome abundance and composition in lung cancer cells through dynamic regulation of histone H3 chaperone systems. Together, we propose that this glucocorticoid-induced nucleosome remodeling establishes a chromatin environment that enables the transition to a mesenchymal-like senescent state in lung cancer cells that favors aggressiveness and poor prognosis. Citation Format: Benito M. Traversa, Devesh Raizada, Eva Kragelj, Julia Spegel, Didem Ilter, Ana P. Gomes. Chronic upregulation of glucocorticoid hormones triggers chromatin remodeling in NSCLC through dynamic regulation of histone H3 chaperones. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4722.

Aging is a main risk factor for cancer. Using human serum we demonstrated that tumor progression and metastases occur, at least in part, as a manifestation of global metabolic deregulation of the aged host. This shows that the role of aging in cancer goes far beyond increased exposure time to mutagens; the aging process coordinates various aspects required for malignancy.

SummaryFrom age 65 onwards, the risk of cancer incidence and associated mortality is substantially higher1-3. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy4. For decades, the link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the well-established role of diet, exercise and small molecules that target the pace of metabolic aging5-8. Here, we show that metabolic alterations that occur with age can render a systemic environment favorable to progression and aggressiveness of tumors. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is significantly up-regulated in the serum of older people, and functions as a mediator of tumor progression. We traced this to the induction of SOX4 and a consequent transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, accumulation of MMA represents a novel link between aging and cancer progression, implicating MMA as a novel therapeutic target for advanced carcinomas.

The risk of cancer and associated mortality increases substantially in humans from the age of 65 years onwards1–6. Nonetheless, our understanding of the complex relationship between age and cancer is still in its infancy2,3,7,8. For decades, this link has largely been attributed to increased exposure time to mutagens in older individuals. However, this view does not account for the established role of diet, exercise and small molecules that target the pace of metabolic ageing9–12. Here we show that metabolic alterations that occur with age can produce a systemic environment that favours the progression and aggressiveness of tumours. Specifically, we show that methylmalonic acid (MMA), a by-product of propionate metabolism, is upregulated in the serum of older people and functions as a mediator of tumour progression. We traced this to the ability of MMA to induce SOX4 expression and consequently to elicit transcriptional reprogramming that can endow cancer cells with aggressive properties. Thus, the accumulation of MMA represents a link between ageing and cancer progression, suggesting that MMA is a promising therapeutic target for advanced carcinomas. Ageing in humans is associated with an increase in circulating methylmalonic acid, which induces expression of SOX4 and promotes tumour progression.

Metastasis is the leading cause of cancer mortality. Chromatin remodeling provides the foundation for the cellular reprogramming necessary to drive metastasis. However, little is known about the nature of this remodeling and its regulation. Here, we show that metastasis-inducing pathways regulate histone chaperones to reduce canonical histone incorporation into chromatin, triggering deposition of H3.3 variant at the promoters of poor-prognosis genes and metastasis-inducing transcription factors. This specific incorporation of H3.3 into chromatin is both necessary and sufficient for the induction of aggressive traits that allow for metastasis formation. Together, our data clearly show incorporation of histone variant H3.3 into chromatin as a major regulator of cell fate during tumorigenesis, and histone chaperones as valuable therapeutic targets for invasive carcinomas.

Growth-promoting signaling molecules, including the mammalian target of rapamycin complex 1 (mTORC1), drive the metabolic reprogramming of cancer cells required to support their biosynthetic needs for rapid growth and proliferation [1]. Glutamine is catabolyzed to α-ketoglutarate (αKG), a tricarboxylic acid (TCA) cycle intermediate, through two deamination reactions, the first requiring glutaminase (GLS) to generate glutamate and the second occurring via glutamate dehydrogenase (GDH) or transaminases [2]. Activation of the mTORC1 pathway has been shown previously to promote the anaplerotic entry of glutamine to the TCA cycle via GDH. Moreover, mTORC1 activation also stimulates the uptake of glutamine, but the mechanism is unknown [3]. It is generally thought that rates of glutamine utilization are limited by mitochondrial uptake via GLS, suggesting that, in addition to GDH, mTORC1 could regulate GLS. Here we demonstrate that mTORC1 positively regulates GLS and glutamine flux through this enzyme. We show that mTORC1 controls GLS levels through the S6K1-dependent regulation of c-Myc (Myc). Molecularly, S6K1 enhances Myc translation efficiency by modulating the phosphorylation of eukaryotic initiation factor eIF4B, which is critical to unwind its structured 5' untranslated region (5'UTR). Finally, our data show that the pharmacological inhibition of GLS is a promising target in pancreatic cancers expressing low levels of PTEN. ispartof: Current biology : CB vol:24 issue:19 pages:2274-80 ispartof: location:England status: published

Lymphangioleiomyomatosis (LAM) is a destructive lung disease of women associated with the metastasis of tuberin-null cells with hyperactive mammalian target of rapamycin complex 1 (mTORC1) activity. Clinical trials with the mTORC1 inhibitor rapamycin have revealed partial efficacy but are not curative. Pregnancy appears to exacerbate LAM, suggesting that estrogen (E 2 ) may play a role in the unique features of LAM. Using a LAM patient-derived cell line (bearing biallelic Tuberin inactivation), we demonstrate that E 2 stimulates a robust and biphasic activation of ERK2 and transcription of the late response-gene Fra1 associated with epithelial-to-mesenchymal transition. In a carefully orchestrated collaboration, activated mTORC1/S6K1 signaling enhances the efficiency of Fra1 translation of Fra1 mRNA transcribed by the E 2 –ERK2 pathway, through the phosphorylation of the S6K1-dependent eukaryotic translation initiation factor 4B. Our results indicate that targeting the E 2 –ERK pathway in combination with the mTORC1 pathway may be an effective combination therapy for LAM.

The acquisition of an invasive phenotype is a critical turning point for malignant tumor cells. CMTM8, a potential tumor suppressor, is frequently down-regulated in solid tumors, and its overexpression induces tumor cell apoptosis. Here, we identify a new role for CMTM8 in regulating tumor cell migration. Reducing CMTM8 expression in HepG2 hepatocellular carcinoma cells results in the acquisition of epithelial-to-mesenchymal transition (EMT) features, including a morphological change from organized epithelial sheets to scattered fibroblast-like shapes, reduction of the epithelial marker E-cadherin, and an increased invasive and migratory ability. These phenotypic changes are mediated in large part by the ERK-MAPK pathway, as the MEK inhibitor U0126 and shRNA-mediated knockdown of ERK2 significantly reversed these phenotypes. Hepatocyte growth factor binding to the c-MET receptor is known to induce EMT in HepG2 cells. We found that CMTM8 knockdown in HepG2 cells induced c-MET signaling and ERK activation. Inhibition of c-MET signaling with the small molecule inhibitor SU11274 or c-MET RNAi blocked the EMT-like changes following CMTM8 knockdown. CMTM8 overexpression in HepG2 cells inhibited hepatocyte growth factor-induced EMT-like morphological changes and cell motility. Down-regulation of CMTM8 also promoted an EMT-like change in MCF-10A cells, indicating a broader role for CMTM8 in regulating cellular transformation.

The alteration of metabolic pathways is a critical strategy for cancer cells to attain the traits necessary for metastasis in disease progression. Here, we find that dysregulation of propionate metabolism produces a pro-aggressive signature in breast and lung cancer cells, increasing their metastatic potential. This occurs through the downregulation of methylmalonyl coenzyme A epimerase (MCEE), mediated by an extracellular signal-regulated kinase 2-driven transcription factor Sp1/early growth response protein 1 transcriptional switch driven by metastatic signalling at its promoter level. The loss of MCEE results in reduced propionate-driven anaplerotic flux and intracellular and intratumoral accumulation of methylmalonic acid, a by-product of propionate metabolism that promotes cancer cell invasiveness. Altogether, we present a previously uncharacterized dysregulation of propionate metabolism as an important contributor to cancer and a valuable potential target in the therapeutic treatment of metastatic carcinomas.