De novo serine biosynthesis from glucose predicts sex-specific response to antifolates in non-small cell lung cancer cell lines

Lung cancer is the leading cause of cancer-related death. Intriguingly, males with non-small cell lung cancer (NSCLC) have a higher mortality rate than females. Here, we investigated the role of serine metabolism as a predictive marker for sensitivity to the antifolate pemetrexed in male and female NSCLC cell lines. Using

Sex differences in cancer mechanisms

We now know that cancer is many different diseases, with great variation even within a single histological subtype. With the current emphasis on developing personalized approaches to cancer treatment, it is astonishing that we have not yet systematically incorporated the biology of sex differences into our paradigms for laboratory and clinical cancer research. While some sex differences in cancer arise through the actions of circulating sex hormones, other sex differences are independent of estrogen, testosterone, or progesterone levels. Instead, these differences are the result of sexual differentiation, a process that involves genetic and epigenetic mechanisms, in addition to acute sex hormone actions. Sexual differentiation begins with fertilization and continues beyond menopause. It affects virtually every body system, resulting in marked sex differences in such areas as growth, lifespan, metabolism, and immunity, all of which can impact on cancer progression, treatment response, and survival. These organismal level differences have correlates at the cellular level, and thus, males and females can fundamentally differ in their protections and vulnerabilities to cancer, from cellular transformation through all stages of progression, spread, and response to treatment. Our goal in this review is to cover some of the robust sex differences that exist in core cancer pathways and to make the case for inclusion of sex as a biological variable in all laboratory and clinical cancer research. We finish with a discussion of lab- and clinic-based experimental design that should be used when testing whether sex matters and the appropriate statistical models to apply in data analysis for rigorous evaluations of potential sex effects. It is our goal to facilitate the evaluation of sex differences in cancer in order to improve outcomes for all patients

Sex differences in brain tumor glutamine metabolism reveal sex-specific vulnerabilities to treatment

BACKGROUND: Brain cancer incidence and mortality rates are greater in males. Understanding the molecular mechanisms that underlie those sex differences could improve treatment strategies. Although sex differences in normal metabolism are well described, it is currently unknown whether they persist in cancerous tissue. METHODS: Using positron emission tomography (PET) imaging and mass spectrometry, we assessed sex differences in glioma metabolism in samples from affected individuals. We assessed the role of glutamine metabolism in male and female murine transformed astrocytes using isotope labeling, metabolic rescue experiments, and pharmacological and genetic perturbations to modulate pathway activity. FINDINGS: We found that male glioblastoma surgical specimens are enriched for amino acid metabolites, including glutamine. Fluoroglutamine PET imaging analyses showed that gliomas in affected male individuals exhibit significantly higher glutamine uptake. These sex differences were well modeled in murine transformed astrocytes, in which male cells imported and metabolized more glutamine and were more sensitive to glutaminase 1 (GLS1) inhibition. The sensitivity to GLS1 inhibition in males was driven by their dependence on glutamine-derived glutamate for α-ketoglutarate synthesis and tricarboxylic acid (TCA) cycle replenishment. Females were resistant to GLS1 inhibition through greater pyruvate carboxylase (PC)-mediated TCA cycle replenishment, and knockdown of PC sensitized females to GLS1 inhibition. CONCLUSION: Our results show that clinically important sex differences exist in targetable elements of metabolism. Recognition of sex-biased metabolism may improve treatments through further laboratory and clinical research. FUNDING: This work was supported by NIH grants, Joshua\u27s Great Things, the Siteman Investment Program, and the Barnard Research Fund

Gonadal sex patterns p21-induced cellular senescence in mouse and human glioblastoma

Males exhibit higher incidence and worse prognosis for the majority of cancers, including glioblastoma (GBM). Disparate survival may be related to sex-biased responses to treatment, including radiation. Using a mouse model of GBM, we show that female cells are more sensitive to radiation, and that senescence represents a major component of the radiation therapeutic response in both sexes. Correlation analyses revealed that the CDK inhibitor p21 and irradiation induced senescence were differentially regulated between male and female cells. Indeed, female cellular senescence was more sensitive to changes in p21 levels, a finding that was observed in wildtype and transformed murine astrocytes, as well as patient-derived GBM cell lines. Using a novel Four Core Genotypes model of GBM, we further show that sex differences in p21-induced senescence are patterned during early development by gonadal sex. These data provide a rationale for the further study of sex differences in radiation response and how senescence might be enhanced for radiation sensitization. The determination that p21 and gonadal sex are required for sex differences in radiation response will serve as a foundation for these future mechanistic studies

Sex Differences in Cancer Metabolism Contribute to Sex-Specific Treatment Responses

Many human diseases show sex differences in incidence, age of onset, and outcome, including autoimmune diseases, neurological diseases, psychiatric disorders, cardiovascular diseases, metabolic diseases, neurodevelopmental disorders, and cancer. For most cancers, incidence and mortality rates are higher in males. Lung and brain cancers are no exception. Overall, lung cancer is more common in males. Furthermore, mortality rates of non-small cell lung cancer (NSCLC), the most common subtype of lung cancer, are significantly higher in males. The underlying reasons for sex differences in NSCLC mortality rates are largely unknown. Most brain cancers are also more common in males. The male prevalence persists across all age groups and all geographical regions, indicating that these sex differences are not solely driven by environmental factors or circulating sex hormones. Glioblastoma (GBM), the most common and aggressive primary brain tumor in adults, exhibits a male to female ratio of 1.6 to 1. Furthermore, female GBM patients have a survival advantage. The biological mechanisms contributing to these sex differences remain unknown. Sex differences in cancer incidence are likely driven by various cellular mechanisms involved in cancer development, such as proliferation, interactions with the immune system, angiogenesis, cell migration, genome instability, and metabolism. Sex differences in cancer survival are likely to involve these mechanisms as well as those that govern treatment response. This dissertation explored sex differences in cancer metabolism and treatment responses in GBM and NSCLC. We show that male and female GBM surgical specimens exhibit considerable sex differences in amino acid metabolite abundance, including glutamine and branched-chain amino acid (BCAA) metabolites. In addition, we show that glutamine uptake is greater in male glioma patients and male murine transformed astrocytes (Nf1-/-; DNp53 astrocytes). Furthermore, we found increased glutamine utilization and glutaminase 1 (GLS1) expression in male transformed astrocytes. Concordantly, GLS1 inhibition leads to greater growth reduction in male transformed astrocytes. The sensitivity to GLS1 inhibition in males is driven by their dependence on glutamine-derived glutamate to replenish their TCA cycle. Female growth is glutamine-independent because of their greater pyruvate carboxylase-mediated TCA cycle replenishment. These findings indicate that male GBM are more dependent on glutamine to maintain proliferation. Metabolite analysis of male and female GBM surgical specimens also showed that BCAA metabolites are more abundant in male GBM. The BCAA leucine stimulates the mTOR pathway, an important regulator of metabolism, growth, and protein biosynthesis. We found that male transformed astrocytes show a greater change in mTOR pathway activity upon serum starvation and pathway stimulation. Furthermore, high phosphorylation levels of mTOR, an indicator for pathway activation, are associated with poor survival in male, but not female, GBM patients. Thus, sex differences in mTOR pathway activity may lead to sex differences in metabolism and vice versa. We also investigated the molecular mechanisms underlying sex differences in other GBM treatment responses. We found that female transformed astrocytes are more sensitive to radiation than male cells. This sex difference in cytotoxicity was driven by a greater induction of senescence in female transformed astrocytes. Furthermore, we identified the CDK inhibitor p21 as a likely mediator of the sex differences in senescence induction. Last, we show that gonadal sex, rather than chromosomal sex, underlie sex differences in p21-mediates senescence response. These findings suggest that sex differences in the senescence response to radiation may contribute to sex differences in treatment response in GBM patients. Last, using publicly available [13C6]glucose tracing data in over 80 NSCLC cell lines, we found that male cells generate significantly more serine from glucose. Furthermore, de novo serine biosynthesis was correlated with antifolate sensitivity in male cells only. Patient mRNA expression data showed that enzymes involved in de novo serine biosynthesis and the folate cycle are higher expressed in male lung and pan-cancer patient samples. Additionally, high serine synthesis gene expression was associated with antifolate sensitivity in male, but not female, patient-derived pan-cancer cell lines. These findings indicate that de novo serine biosynthesis is a reliable marker for antifolate sensitivity in male NSCLC in vitro cultures. In summary, these data show that sex differences in metabolism persist in GBM and NSCLC and that these sex differences may be utilized to develop and improve treatment approaches to enhance outcome for both, male and female cancer patients

Cell-intrinsic, Bmal1-dependent Circadian Regulation of Temozolomide Sensitivity in Glioblastoma

The safety and efficacy of chemotherapeutics can vary as a function of the time of their delivery during the day. This study aimed to improve the treatment of glioblastoma (GBM), the most common brain cancer, by testing whether the efficacy of the DNA alkylator temozolomide (TMZ) varies with the time of its administration. We found cell-intrinsic, daily rhythms in both human and mouse GBM cells. Circadian time of treatment affected TMZ sensitivity of murine GBM tumor cells in vitro. The maximum TMZ-induced DNA damage response, activation of apoptosis, and growth inhibition occurred near the daily peak in expression of the core clock gene Bmal1. Deletion of Bmal1 (Arntl) abolished circadian rhythms in gene expression and TMZ-induced activation of apoptosis and growth inhibition. These data indicate that tumor cell-intrinsic circadian rhythms are common to GBM tumors and can regulate TMZ cytotoxicity. Optimization of GBM treatment by timing TMZ administration to daily rhythms should be evaluated in prospective clinical trials.Fil: Slat, Emily A.. Washington University in St. Louis; Estados UnidosFil: Sponagel, Jasmin. Washington University in St. Louis; Estados UnidosFil: Marpegan, Luciano. Washington University in St. Louis; Estados Unidos. Universidad Nacional de Quilmes; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Simon, Tatiana. Washington University in St. Louis; Estados UnidosFil: Kfoury, Najla. Washington University in St. Louis; Estados UnidosFil: Kim, Albert. Washington University in St. Louis; Estados UnidosFil: Binz, Andrea. Washington University in St. Louis; Estados UnidosFil: Herzog, Erik D.. Washington University in St. Louis; Estados UnidosFil: Rubin, Joshua B.. Washington University in St. Louis; Estados Unido

Dependence of Intracellular and Exosomal microRNAs on Viral <i>E6/E7</i> Oncogene Expression in HPV-positive Tumor Cells

<div><p>Specific types of human papillomaviruses (HPVs) cause cervical cancer. Cervical cancers exhibit aberrant cellular microRNA (miRNA) expression patterns. By genome-wide analyses, we investigate whether the intracellular and exosomal miRNA compositions of HPV-positive cancer cells are dependent on endogenous <i>E6/E7</i> oncogene expression. Deep sequencing studies combined with qRT-PCR analyses show that <i>E6/E7</i> silencing significantly affects ten of the 52 most abundant intracellular miRNAs in HPV18-positive HeLa cells, downregulating miR-17-5p, miR-186-5p, miR-378a-3p, miR-378f, miR-629-5p and miR-7-5p, and upregulating miR-143-3p, miR-23a-3p, miR-23b-3p and miR-27b-3p. The effects of <i>E6/E7</i> silencing on miRNA levels are mainly not dependent on p53 and similarly observed in HPV16-positive SiHa cells. The <i>E6/E7</i>-regulated miRNAs are enriched for species involved in the control of cell proliferation, senescence and apoptosis, suggesting that they contribute to the growth of HPV-positive cancer cells. Consistently, we show that sustained <i>E6/E7</i> expression is required to maintain the intracellular levels of members of the miR-17~92 cluster, which reduce expression of the anti-proliferative <i>p21</i> gene in HPV-positive cancer cells. In exosomes secreted by HeLa cells, a distinct seven-miRNA-signature was identified among the most abundant miRNAs, with significant downregulation of let-7d-5p, miR-20a-5p, miR-378a-3p, miR-423-3p, miR-7-5p, miR-92a-3p and upregulation of miR-21-5p, upon <i>E6/E7</i> silencing. Several of the <i>E6/E7</i>-dependent exosomal miRNAs have also been linked to the control of cell proliferation and apoptosis. This study represents the first global analysis of intracellular and exosomal miRNAs and shows that viral oncogene expression affects the abundance of multiple miRNAs likely contributing to the <i>E6/E7</i>-dependent growth of HPV-positive cancer cells.</p></div

HPV oncogenes control <i>p21</i> expression at multiple levels.

<p>E6 can repress <i>p21</i> transcription at the promoter level by inducing the degradation of the <i>p21</i> transcriptional activator p53; sustained <i>E6/E7</i> expression maintains the concentration of miR-17 family members in HPV-positive cancer cells which repress <i>p21</i> expression by targeting the <i>p21</i> mRNA; the E7 protein can directly bind to the p21 protein and inhibit its function.</p

Inhibition of endogenous HPV16 <i>E6/E7</i> expression: Effects on selected intracellular miRNAs.

<p><b>(A)</b> Immunoblot analysis of HPV16 E7, HPV16 E6, p53 and p21 protein levels, 72 h after transfection of SiHa cells with si16E6/E7 or control siRNA (siContr-1), or upon mock treatment. α-Tubulin: loading control. <b>(B)</b> qRT-PCR analyses of ten selected cellular miRNAs, 72 h after transfection of SiHa cells with si16E6/E7 or siContr-1. Cellular miRNA levels were normalized to the snRNA <i>RNU6–2</i> and calculated relative to siContr-1 (log₂ display). Dashed lines: 1.5-fold up- or downregulation (log₂(1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*) and p ≤ 0.01 (**)).</p

Effects of the p53 status on the <i>E6/E7</i>-dependent modulation of intracellular miRNAs.

<p><b>(A)</b> qRT-PCR analysis of HPV18 <i>E6/E7</i> (left panel) and <i>p21</i> (right panel) mRNA expression, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7, control siRNA (siContr-1), or upon mock treatment. mRNA levels were normalized to <i>ACTB</i> and calculated relative to the mock control (mock). Data represent mean ± SEM (n = 3). Asterisks above columns indicate statistically significant differences from siContr-1-treated cells (p ≤ 0.05 (*), p ≤ 0.001 (***)). <b>(B)</b> Immunoblot analysis of HPV18 E6, p53 and p21 protein levels, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1, or upon mock treatment. α-Tubulin: loading control. <b>(C)</b> qRT-PCR analyses of selected cellular miRNAs, 72 h after transfection of parental or “p53-null” HeLa cells with si18E6/E7 or siContr-1. miR-34a-3p, positive control miRNA (p53-inducible). Cellular miRNA levels were normalized to snRNA <i>RNU6–2</i> and calculated relative to siContr-1 (log₂ display). Dashed lines: 1.5-fold up- or downregulation (log₂(1.5) = 0.585). Data represent mean ± SEM (n = 3). Asterisks indicate statistically significant differences (p ≤ 0.05 (*), p ≤ 0.01 (**) and p ≤ 0.001 (***)).</p