Supplementary Figure from Pyruvate Kinase M1 Suppresses Development and Progression of Prostate Adenocarcinoma

Fold change in the expression of HPV genes in SCC090 cells after 5-aza treatment, as determined by qRT-PCR

Figure 1: Oxygen consumption rate of prostate cancer cell lines with different concentrations of metformin. Error bars depict the standard deviation. Standard deviation and p-values are calculated from three biological replicates. Figure 2: Glucose (upper panel) and reductive glutamine (lower panel) contribution to fatty acids in prostate cancer cell lines treated with metformin fitted from the measured incorporation of U-13C labeled glucose or 5-13C labeled glutamine (reductive glutamine contribution (red)) into palmitate. Error bars depict the 95 % confidence interval. Confidence intervals and p-values are calculated from two biological replicates. Figure 3: Comparison of glutamine contribution to tricarboxylic acid cycle in prostate cancer cell lines treated with metformin or rotenone measured by the incorporation of U-13C labeled glutamine into alpha-ketoglutarate (upper panel) and reductive glutamine contribution to palmitate using 5-13C labeled glutamine (lower panel). Confidence intervals and p-values are calculated from two biological replicates. Figure 4: (A) Relative cell count of prostate cancer cell lines treated with a combination of metformin and the glutaminase inhibitor BPTES. Cell counts were normalized to the condition with no metformin and no BPTES added. (B) Relative cell count of Huh7 liver cancer cells, which can grow without glutamine in the presence or absence of metformin. Cell counts were normalized to the corresponding condition with no metformin added. Error bars depict the standard deviation. Standard deviation and p-values are calculated from three biological replicates. Figure 5: (A) TSC2 expression in TSC2 knockdown cells and control. Standard diviation is calculated from three technical replicates. p-values are < 0.005.(B) Metformin sensitivity (2.5mM) in LNCaP cells with and without dimethyl alpha-ketoglutarate given by cell counts normalized to the corresponding condition with no metformin. Standard deviation and p-values are calculated from at least three biological replicates. p-value is < 0.05. All error bars depict the standard deviation.

Abstract Altered metabolism helps sustain cancer cell proliferation and survival. Most cancers, including prostate cancers, express the M2 splice isoform of pyruvate kinase (PKM2), which can support anabolic metabolism to support cell proliferation. However, Pkm2 expression is dispensable for the formation and growth of many cancers in vivo. Expression of pyruvate kinase isoform M1 (Pkm1) is restricted to relatively few tissues and has been reported to promote growth of select tumors, but the role of PKM1 in cancer has been less studied than PKM2. To test how differential expression of pyruvate kinase isoforms affects cancer initiation and progression, we generated mice harboring a conditional allele of Pkm1 and crossed these mice, or those with a Pkm2 conditional allele, with a Pten loss-driven prostate cancer model. Pkm1 loss led to increased PKM2 expression and accelerated prostate cancer development, whereas Pkm2 deletion led to increased PKM1 expression and suppressed tumor progression. Metabolic profiling revealed altered nucleotide levels in tumors with high PKM1 expression, and failure of these tumors to progress was associated with DNA replication stress and senescence. Consistent with these data, a small molecule pyruvate kinase activator that mimics a high activity PKM1-like state suppressed progression of established prostate tumors. Analysis of human specimens showed PKM2 expression is retained in most human prostate cancers. Overall, this study uncovers a role for pyruvate kinase isoforms in prostate cancer initiation and progression, and argues that pharmacologic pyruvate kinase activation may be beneficial for treating prostate cancer. Significance: Differential expression of PKM1 and PKM2 impacts prostate tumorigenesis and suggests a potential therapeutic vulnerability in prostate cancer.

Abstract Background: As the incidence of human papillomavirus (HPV)-related head and neck squamous cell carcinoma (HNSCC) continues to rise, mechanistic knowledge of HPV16-driven HNSCC remains incomplete. HPV(+) and HPV(-) HNSCCs are molecularly distinct, with altered signaling pathways that have been characterized by proteomic technologies. We recently reported that poly(ADP-ribose)polymerase-1 (PARP-1) is overexpressed in HPV(+) compared to HPV(-) HNSCC. PARP-1 is a multi-functional protein with the ability to produce polymers of ADP-ribose attached to proteins and with major known roles in maintaining genomic integrity, DNA damage repair, transcription, DNA replication, and cell cycle regulation. However, PARP-1 functions in HPV infection and in head and neck cancer have not been fully uncovered. Here, we examine the role of PARP-1 in HPV infection, the interactions of HPV with cellular proteins, and the therapeutic implications of PARP-1 inhibition in HPV(+) HNSCC. Methods: PARP activity was assessed by immunoblotting for levels of poly(ADP-ribose) and by enzymatic PARP-1 ELISA. Expression of HPV genes was measured by RT-PCR. Murine kidney epithelial cells were established from PARP-1 wild-type and knockout mice, and the absence of PARP-1 expression was confirmed by RT-PCR and immunoblotting. Infection with HPV pseudovirus (PsV) containing a GFP expression plasmid was detected by L1 immunofluorescence staining and GFP detection. Survival studies were done in vitro and in vivo with PARP inhibitors veliparib and olaparib. Results: In addition to being overexpressed, we found that PARP-1 was highly enzymatically active in HPV(+) HNSCC as compared to HPV(-) tumors. HPV16 PsV and major capsid protein L1 activated PARP-1 in vitro and in cells. Depletion of PARP-1 and chemical PARP inhibition repressed HPV16 cellular entry, and infection of cells with HPV PsV increased sensitivity of HPV(-) cells to PARP inhibition. Conclusions: We identified a functional role of PARP-1 in initial HPV infection and in HPV-associated cancer. PARP-1 is activated by HPV major capsid protein L1, and HPV(+) HNSCCs maintain PARP-1 enzymatic activity. On top of its role in initial HPV infection, PARP-1 also appears to be important for survival of HPV-infected cells, as inhibition of PARP-1 sensitized cells to HPV PsV infection. These findings suggest that PARP inhibition may hold significant therapeutic potential in the treatment of HPV-associated HNSCC, with our work resulting in the initiation of a window clinical trial ongoing at the Yale Cancer Center. Citation Format: Cassie Pan, Asel Biktasova, Michael Hajek, Andrew Sewell, Tejas Sathe, Gary Bellinger, Wendell Yarbrough, Natalia Issaeva. PARP-1 supports HPV infection and HPV-associated head and neck cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-333.

Abstract Purpose: DNA methylation in human papillomavirus–associated (HPV+) head and neck squamous cell carcinoma (HNSCC) may have importance for continuous expression of HPV oncogenes, tumor cell proliferation, and survival. Here, we determined activity of a global DNA-demethylating agent, 5-azacytidine (5-aza), against HPV+ HNSCC in preclinical models and explored it as a targeted therapy in a window trial enrolling patients with HPV+ HNSCC. Experimental Design: Sensitivity of HNSCC cells to 5-aza treatment was determined, and then 5-aza activity was tested in vivo using xenografted tumors in a mouse model. Finally, tumor samples from patients enrolled in a window clinical trial were analyzed to identify activity of 5-aza therapy in patients with HPV+ HNSCC. Results: Clinical trial and experimental data show that 5-aza induced growth inhibition and cell death in HPV+ HNSCC. 5-aza reduced expression of HPV genes, stabilized p53, and induced p53-dependent apoptosis in HNSCC cells and tumors. 5-aza repressed expression and activity of matrix metalloproteinases (MMP) in HPV+ HNSCC, activated IFN response in some HPV+ head and neck cancer cells, and inhibited the ability of HPV+ xenografted tumors to invade mouse blood vessels. Conclusions: 5-aza may provide effective therapy for HPV-associated HNSCC as an alternative or complement to standard cytotoxic therapy. Clin Cancer Res; 23(23); 7276–87. ©2017 AACR.

We previously reported that combining a phosphoinositide 3-kinase (PI3K) inhibitor with a poly-ADP Rib polymerase (PARP)-inhibitor enhanced DNA damage and cell death in breast cancers that have genetic aberrations in BRCA1 and TP53. Here, we show that enhanced DNA damage induced by PI3K inhibitors in this mutational background is a consequence of impaired production of nucleotides needed for DNA synthesis and DNA repair. Inhibition of PI3K causes a reduction in all four nucleotide triphosphates, whereas inhibition of the protein kinase AKT is less effective than inhibition of PI3K in suppressing nucleotide synthesis and inducing DNA damage. Carbon flux studies reveal that PI3K inhibition disproportionately affects the nonoxidative pentose phosphate pathway that delivers Rib-5-phosphate required for base ribosylation. In vivo in a mouse model of BRCA1-linked triple-negative breast cancer (K14-Cre BRCA1(f/f)p53(f/f)), the PI3K inhibitor BKM120 led to a precipitous drop in DNA synthesis within 8 h of drug treatment, whereas DNA synthesis in normal tissues was less affected. In this mouse model, combined PI3K and PARP inhibition was superior to either agent alone to induce durable remissions of established tumors.

Radiation and chemotherapy that are commonly used to treat human cancers damage cellular DNA. DNA damage appears to be more toxic to cancer cells than normal cells, most likely due to deregulated checkpoint activation and/or deficiency in DNA repair pathways that are characteristics of many tumors. However, unwanted side effects arise as a result of DNA damage to normal cells during the treatment.Here, we show that roscovitine, a cyclin-dependent kinase (CDK) inhibitor that inhibits CDK-1, CDK-2, CDK-5, CDK-7, and CDK-9 due to competitive binding to the ATP site on the kinases, causes significant DNA damage followed by p53-dependent cell death in human papilloma virus (HPV)-positive, but not in HPV-negative, head and neck cancer cells. Since HPV positivity was a molecular marker for increased sensitivity of cells to roscovitine, we reasoned that systemic roscovitine administration would not be toxic to healthy HPV-negative tissue. Indeed, low roscovitine doses significantly inhibited the growth of HPV-associated xenografted tumors in mice without causing any detectable side effects.Given that inhibition of CDKs has been shown to inhibit replication of several viruses, we suggest that roscovitine treatment may represent a selective and safe targeted therapeutic option against HPV-positive head and neck cancer.

United States. Dept. of Defense. Congressionally Directed Medical Research Programs (Postdoctoral Award W81XWH-12-1-0466)

Metabolic regulation influences cell proliferation. The influence of pyruvate kinase isoforms on tumor cells has been extensively studied, but whether PKM2 is required for normal cell proliferation is unknown. We examine how PKM2 deletion affects proliferation and metabolism in nontransformed, nonimmortalized PKM2-expressing primary cells. We find that deletion of PKM2 in primary cells results in PKM1 expression and proliferation arrest. PKM1 expression, rather than PKM2 loss, is responsible for this effect, and proliferation arrest cannot be explained by cell differentiation, senescence, death, changes in gene expression, or prevention of cell growth. Instead, PKM1 expression impairs nucleotide production and the ability to synthesize DNA and progress through the cell cycle. Nucleotide biosynthesis is limiting, as proliferation arrest is characterized by severe thymidine depletion, and supplying exogenous thymine rescues both nucleotide levels and cell proliferation. Thus, PKM1 expression promotes a metabolic state that is unable to support DNA synthesis.

Abstract Metformin inhibits cancer cell proliferation, and epidemiology studies suggest an association with increased survival in patients with cancer taking metformin; however, the mechanism by which metformin improves cancer outcomes remains controversial. To explore how metformin might directly affect cancer cells, we analyzed how metformin altered the metabolism of prostate cancer cells and tumors. We found that metformin decreased glucose oxidation and increased dependency on reductive glutamine metabolism in both cancer cell lines and in a mouse model of prostate cancer. Inhibition of glutamine anaplerosis in the presence of metformin further attenuated proliferation, whereas increasing glutamine metabolism rescued the proliferative defect induced by metformin. These data suggest that interfering with glutamine may synergize with metformin to improve outcomes in patients with prostate cancer. Cancer Res; 73(14); 4429–38. ©2013 AACR.

Abstract CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well defined. Studies from our laboratory revealed a biochemical pathway by which CDCP1 participates in the activation of Src-family kinase (SFK) members and the coupling of SFK-activation to the phosphorylation and regulation of protein kinase C-delta (PKC-δ). Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in a HIF-2α, but not HIF-1α, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, while overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation, as well as, significantly promotes lung metastases in NOD/SCID mice. To investigate the relationship between HIF-2α and CDCP1 expression, we performed a correlation analysis in the largest up-to-date collection (Sanger Cell Line Project) of cancer cell line microarray data (n=732). We found a dramatic concordance in the expression of HIF-2α and CDCP1 (Pearson's correlation, P <1x10-20), indicating that cancers with high HIF-2α expression tend to have high levels of CDCP1 expression. We next asked whether other known HIF-2α target genes also correlate in this expression analysis. Remarkably, MET and EGFR, which are hypoxia regulated and known HIF-2α target genes, also displayed a strong correlation with HIF-2α and CDCP1 expression. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma (ccRCC) shows that increased CDCP1 expression correlates with decreased overall survival. Interestingly, high-grade ccRCCs (G3, G4) expressed significantly higher (P = 0.03, t-test) levels of CDCP1 protein compared to lower grade tumors (G1, G2), suggesting that CDCP1 expression increases progressively with higher ccRCC tumor grade. Furthermore, hypoxia activates Src signaling and the Src inhibitor (Dasatinib) prevents the hypoxia-induced phosphorylation of CDCP1. Thereby, reinforcing that CDCP1 is an SFK-associated receptor, which promotes migration and metastasis and suggests that hypoxia-induced CDCP1 signaling may further stimulate a more aggressive cancer phenotype. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that therapeutic approaches targeting CDCP1, such as monoclonal antibodies, could be beneficial in the treatment of metastatic cancers. Supported by NIH grant 5R01GM056203-15 to L.C.C and Dana Farber/Harvard Cancer Center Career Development Award to B.M.E. Citation Format: Brooke M. Emerling, Cyril Benes, Eric Bell, George Poulogiannis, Kevin Courtney, Hui Lui, Rayman Choo-Wing, Gary Bellinger, Stephen Soltoff, Lewis Cantley. Identification of CDCP1 as a HIF-2α target gene involved in the regulation of cancer cell migration and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2013-4588

CUB domain-containing protein 1 (CDCP1) is a transmembrane protein that is highly expressed in stem cells and frequently overexpressed and tyrosine-phosphorylated in cancer. CDCP1 promotes cancer cell metastasis. However, the mechanisms that regulate CDCP1 are not well-defined. Here we show that hypoxia induces CDCP1 expression and tyrosine phosphorylation in hypoxia-inducible factor (HIF)-2α–, but not HIF-1α–, dependent fashion. shRNA knockdown of CDCP1 impairs cancer cell migration under hypoxic conditions, whereas overexpression of HIF-2α promotes the growth of tumor xenografts in association with enhanced CDCP1 expression and tyrosine phosphorylation. Immunohistochemistry analysis of tissue microarray samples from tumors of patients with clear cell renal cell carcinoma shows that increased CDCP1 expression correlates with decreased overall survival. Together, these data support a critical role for CDCP1 as a unique HIF-2α target gene involved in the regulation of cancer metastasis, and suggest that CDCP1 is a biomarker and potential therapeutic target for metastatic cancers.

Thioredoxin-interacting protein (TXNIP) is an α-arrestin family protein that is induced in response to glucose elevation. It has been shown to provide a negative feedback loop to regulate glucose uptake into cells, though the biochemical mechanism of action has been obscure. Here, we report that TXNIP suppresses glucose uptake directly, by binding to the glucose transporter GLUT1 and inducing GLUT1 internalization through clathrin-coated pits, as well as indirectly, by reducing the level of GLUT1 messenger RNA (mRNA). In addition, we show that energy stress results in the phosphorylation of TXNIP by AMP-dependent protein kinase (AMPK), leading to its rapid degradation. This suppression of TXNIP results in an acute increase in GLUT1 function and an increase in GLUT1 mRNA (hence the total protein levels) for long-term adaptation. The glucose influx through GLUT1 restores ATP-to-ADP ratios in the short run and ultimately induces TXNIP protein production to suppress glucose uptake once energy homeostasis is reestablished.

The pyruvate kinase M2 isoform (PKM2) is expressed in cancer and plays a role in regulating anabolic metabolism. To determine whether PKM2 is required for tumor formation or growth, we generated mice with a conditional allele that abolishes PKM2 expression without disrupting PKM1 expression. PKM2 deletion accelerated mammary tumor formation in a Brca1-loss-driven model of breast cancer. PKM2 null tumors displayed heterogeneous PKM1 expression, with PKM1 found in nonproliferating tumor cells and no detectable pyruvate kinase expression in proliferating cells. This suggests that PKM2 is not necessary for tumor cell proliferation and implies that the inactive state of PKM2 is associated with the proliferating cell population within tumors, whereas nonproliferating tumor cells require active pyruvate kinase. Consistent with these findings, variable PKM2 expression and heterozygous PKM2 mutations are found in human tumors. These data suggest that regulation of PKM2 activity supports the different metabolic requirements of proliferating and nonproliferating tumor cells.

Abstract The PTEN and p53 tumor suppressors are the most commonly altered genes in human cancer, including prostate cancer (PCa). Loss of PTEN is associated with increased Gleason score and clinical recurrence, and the majority of human metastatic PCas have PTEN loss via multiple mechanisms. Mice with prostate-specific homozygous deletion of PTEN develop invasive PCa albeit with prolonged latency of 6-8 months. Combined PTEN/p53 inactivation in mouse prostate elicits invasive cancer by 9 weeks of age and invariable lethality by 6 months of age. Since PTEN loss results in PI3K/mTOR pathway activation, we evaluated the impact of GSK458 (PI3K/mTOR inhibitor) and GSK418 (PI3K beta/delta isoform-specific inhibitor), singly and in combination with GSK212 (MEK inhibitor), in uncastrated, prostate-specific PTEN/p53 double knockout mice (4-6 months) and PTEN mice (11-14 months), respectively, harboring advanced PCa. The drugs were administered by daily oral gavage for 3 weeks with serial 18FDG-PET/MRI imaging at baseline, 2 days, 1 week, 2 weeks and 3 weeks post-treatment respectively. GSK458 treatment of PTEN/p53 and PTEN mice results in reduction in FDG-PET uptake as early as 24 h post-treatment, with 40% tumor shrinkage by 1 week post-treatment, but rapid regrowth of 18FDG-avid tumor by 2-3 weeks post-treatment. This acquired resistance was found to be mediated in part through upregulation of multiple receptor tyrosine kinases. In contrast, PTEN/p53 and PTEN mice did not respond to GSK418, by either FDG-PET or MRI analysis. We observed increased pERK/total ERK ratio by Western blot analysis and increased p-ERK staining by immunohistochemistry in GSK458 and GSK418-treated PTEN/53 mice, respectively. Treatment of PTEN/p53 and PTEN mice with GSK212 resulted in an approx. 40% reduction in tumor volume over 3 weeks. Treatment of PTEN/p53 mice with a combination of GSK458 plus GSK212 resulted in approx. 60% reduction of tumor volume at 3 week post-treatment. Strikingly, treatment of prostate-specific PTEN only mice with GSK458 plus GSK212 combination resulted in a >90% tumor regression at 3 weeks post-treatment. These results demonstrate the potential utlitity of PI3K/MEK-directed combination therapies in the neoadjuvant setting for locally advanced disease or hormone-sensitive phase in metastatic disease, thus delaying the need for hormone ablative therapy and its associated morbidity in advanced PCa. The data underscore the value of genetically engineered mouse models to co-clinically evaluate biomarkers of response and resistance to targeted therapies, and elucidate mechanisms of acquired resistance early in clinical development. The design of “personalized” combination therapies to overcome resistance to PI3K/MEK-directed therapies in the hormone-naive and castration-resistant contexts are currently underway in multiple GEMMs and Phase Ib co-clinical trials in advanced PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-365. doi:1538-7445.AM2012-LB-365