**6. Pathways affected by SCNAs in serous ovarian cancer**

Each cancer probably evolves at least 6–10 independent oncogene or tumor suppressor alterations [69] to circumvent natural homeostatic controls known as the "Hallmarks of Cancer" [70, 71]. These hallmarks include the evasion of regulated cell death, immortalization through telomere maintenance, defects in cell cycle control, immune system suppression, and enabling of metastatic capacity through physical and metabolic means. Traditionally, it has been assumed that single gene mutations are responsible for many of these oncogenic changes. Altered p53 function promotes escape to half of these hallmarks on its own, and mutations in strong oncogenes such as Ras family members, or growth factor receptor genes such as FGFRs, Her2 and even Met supplement many of the remaining hallmarks.

Individual gene amplifications can impact serous ovarian cancer. Aside from *TP53* and *BRCA1/2* mutations already discussed, tumors appear to be selected for specific chromosomal aberrations. Amplification of chromosomal region 8q, which contains the oncogenes *MYC* and *PTK2*, is a commonly found SCNV is SOC Myc overexpression promotes cell cycle progression, angiogenesis, and expression of target genes downstream of many other oncogenic factors such as NF-kB, β-catenin, and growth factor receptors [72]. Myc activation coordinately drives proliferation and promotes apoptosis, though since Myc-mediated apoptosis is p53-dependent, the pathway is averted. *PTK2*, the gene encoding focal adhesion kinase (FAK), enables metastatic phenotypes, cancer stem cell self-renewal, and neovascularization [73]. It is often co-amplified with *MYC*, as they both lie within cytoband 8q24. Myc overexpression is difficult to target therapeutically, although there are clinical trials underway for FAK inhibitors [74].

Recently, we analyzed single nucleotide and short 'in frame' deletion mutations across 120 validated oncogenes and tumor suppressors, finding that as many as 48% of serous ovarian primary tumors do not contain mutations in *any known* tumor suppressor or oncogene other than *TP53* [8]. Nonetheless, the average tumor has two-thirds of its genome altered by SCNAs. These findings support the notion that the actions of single oncogenes and tumor suppressors can only explain a portion of the genetics of ovarian cancer.

To analyze this, we developed new pathway network analytics tools to identify disrupted pathways in serous ovarian cancer in this unusually unstable genetic background. Despite the high levels of heterogeneity across patients, we found that coincident gene disruptions fell along surprisingly consistent patterns tumor-to-tumor, specifically suppressing or amplifying specific cellular pathways.

#### **6.1. Autophagy**

By far the most significantly suppressed pathway which stood out as unique in serous ovarian cancer and triple negative breast cancer was macroautophagy, which is most commonly known, simply, as autophagy. The term autophagy ("*self-eating*") appropriately defines the process that cells use to recycle various macromolecular components, such as protein aggregates, lipids, and even entire organelles [75]. Autophagy is a primary method for the cellular catabolism, complementing turnover of proteins by the ubiquitin-proteosome system. Autophagy is described in terms of flux, which is the throughput of cellular '*detritus'* into autophagosomes, their transport to lysosomes, and subsequent enzymatic digestion. Ovarian cancer autophagy deletions impact the process primarily through deletions in *BECN1* (>75% of serous ovarian cancers) and in *MAP1LC3B* (>80% of serous ovarian cancers), though other genes are frequently affected. The average SOC tumor is 1 N across at least five different alleles; 95% of all serous ovarian cancers are deleted in BECN1 or *MAP1LC3B* and two others. The deficiency is as characteristic of SOC as p53 mutation. In addition to their roles in regulating chromosomal instability outlined above, *MAP1LC3B* and BECN1 (with the class three PI3 Kinase VPS34) play key roles in the formation of the early autophagosome, the phagophore, and recruitment autophagosome expansion proteins [76, 77].

Given this critical cellular function, we considered it counter-intuitive that cancer cells would delete a wide array of autophagy genes. In fact, KRAS mutant cancers have been described as "addicted" to autophagy, particularly in hypoxic or otherwise nutrient-stressed microenvironments [78]. This interpretation has been debated [79, 80], but the fact that autophagy has been established as a tumor suppressor system [81, 82], it is not exclusive of the possibility that specific tumor genotypes can promote addiction to autophagy [78]. Mono-allelic losses in the autophagy gene *BECN1* (homozygous deletions occur in only 0.9% of SOC cases) potentiate early development of tumors in mouse models [83, 84]. In this context, it is not at all counterintuitive to consider that these gene losses likely synergize with defects in the BRCA1/2 pathway, the p53 pathway, and other initial SCNAs, thus producing the unique extreme level of aneuploidy associated with SOC. Moreover, the loss of gene copies does not completely "turnoff" autophagy. In fact, ovarian tumor cells, like other cells, require autophagy to provide clearing of protein aggregates, metabolic byproducts (especially in hypoxic environments), and possibly even to permit cell division, given their aneuploid state and relative chromosomal instability. This, in turn, may provide a second selection criteria for depressed autophagy. Autophagy is induced by missegregating chromosomes, chromosomal instability is a hallmark of SOC, and extreme induction of autophagy can promote cell death [85]. Therefore, it may be more appropriate to use the term "disrupted" rather than "suppressed" to define how ovarian cancer autophagy varies from that seen in normal somatic cells. The state renders SOC sensitive to agents that perturb autophagy by inhibiting the autophagic flux, or via the creation of proteotoxic stresses which must be resolved by autophagy (as discussed below).

#### **6.2. Proteosome**

In summary, *BRCA1/2* and homologous repair components are often suppressed by genetic deletions in SOC. This leads to further increases in SCNA formation and potentially independent metastatic phenotypes. However, SOC patients may benefit from the cancer's reliance on

Each cancer probably evolves at least 6–10 independent oncogene or tumor suppressor alterations [69] to circumvent natural homeostatic controls known as the "Hallmarks of Cancer" [70, 71]. These hallmarks include the evasion of regulated cell death, immortalization through telomere maintenance, defects in cell cycle control, immune system suppression, and enabling of metastatic capacity through physical and metabolic means. Traditionally, it has been assumed that single gene mutations are responsible for many of these oncogenic changes. Altered p53 function promotes escape to half of these hallmarks on its own, and mutations in strong oncogenes such as Ras family members, or growth factor receptor genes such as

Individual gene amplifications can impact serous ovarian cancer. Aside from *TP53* and *BRCA1/2* mutations already discussed, tumors appear to be selected for specific chromosomal aberrations. Amplification of chromosomal region 8q, which contains the oncogenes *MYC* and *PTK2*, is a commonly found SCNV is SOC Myc overexpression promotes cell cycle progression, angiogenesis, and expression of target genes downstream of many other oncogenic factors such as NF-kB, β-catenin, and growth factor receptors [72]. Myc activation coordinately drives proliferation and promotes apoptosis, though since Myc-mediated apoptosis is p53-dependent, the pathway is averted. *PTK2*, the gene encoding focal adhesion kinase (FAK), enables metastatic phenotypes, cancer stem cell self-renewal, and neovascularization [73]. It is often co-amplified with *MYC*, as they both lie within cytoband 8q24. Myc overexpression is difficult to target thera-

Recently, we analyzed single nucleotide and short 'in frame' deletion mutations across 120 validated oncogenes and tumor suppressors, finding that as many as 48% of serous ovarian primary tumors do not contain mutations in *any known* tumor suppressor or oncogene other than *TP53* [8]. Nonetheless, the average tumor has two-thirds of its genome altered by SCNAs. These findings support the notion that the actions of single oncogenes and tumor

To analyze this, we developed new pathway network analytics tools to identify disrupted pathways in serous ovarian cancer in this unusually unstable genetic background. Despite the high levels of heterogeneity across patients, we found that coincident gene disruptions fell along surprisingly consistent patterns tumor-to-tumor, specifically suppressing or amplifying

By far the most significantly suppressed pathway which stood out as unique in serous ovarian cancer and triple negative breast cancer was macroautophagy, which is most commonly

DNA repair pathways, as inhibition of PARPs prolongs progression free survival.

**6. Pathways affected by SCNAs in serous ovarian cancer**

120 Ovarian Cancer - From Pathogenesis to Treatment

FGFRs, Her2 and even Met supplement many of the remaining hallmarks.

peutically, although there are clinical trials underway for FAK inhibitors [74].

suppressors can only explain a portion of the genetics of ovarian cancer.

specific cellular pathways.

**6.1. Autophagy**

Interestingly, a number of other proteostasis control pathways were suppressed in serous ovarian cancer, and foremost among these is complementary to autophagy, the ubiquitinproteasome system. The core subunits, encoded by *PSMA1*, *PSMB1*, and *PSMC1*, are monoallelically deleted in 49, 62, and 41% of patients, respectively. Interestingly, the most interactive and deleted components of the proteasomal degradation pathway in ovarian cancer are enriched for cell cycle control related E3 ligases, including Park2, Fzr1, and Ube2d3. This suggests that not only is the core recycling process partly compromised by the core component deletions, but that the pathway is redirected to allow for cell cycle progression proteins to persist and push the cell through division. The latter finding is perhaps to be expected, given that this has been established as a mechanism for tumor formation in many reviews [86–88]. Yet the proteasome may have a similar function to autophagy in suppressing aneuploidy. In a screen for mutations which are enabling for cell cycle progression in aneuploidy cells, ubiquitin-proteasomal degradation components were a top hit [89].

an essential reaction for the formation of adipose tissue. The pathway is further reinforced by overexpression of *LPIN1* and *LPIN3* genes. While targeting metabolism has not historically been successful in cancer treatment, overexpression of these genes may act as early identifiers

Genomic Copy Number Alterations in Serous Ovarian Cancer

http://dx.doi.org/10.5772/intechopen.72695

123

An unusually altered pathway in serous ovarian cancer bridges metabolism, fatty acid oxidation and proteostasis disruption: *peroxisome* biogenesis. Peroxisomes are subcellular organelles whose primary function is to metabolize reactive-oxygen species and provide lipids to other organelles [101]. This pathway is amplified in serous ovarian cancer, and lung adenocarcinoma only [8]. *PEX5*, *PEX5L*, and *PEX19* are all commonly amplified. Pex5 and Pex19 bind to peroxisome enzymes in the cytosol and direct them to the peroxisome matrix [102, 103]. In fact, amplification of PEX5 is associated with poorer outcome in SOC. Peroxiredoxin-1 is also strongly amplified, and can be detected at increased levels in ovarian cancer patients' serum [104], and is also associated with lung cancer malignancy [105]. Upregulation of this pathway, and those associated with phospholipid metabolism may provide to a means to overcome oxidative stress, perform fatty acid beta-oxidation, and resist lipotoxicity associated

While each of these pathways can help to define phenotypes associated with SOC, they also have the capacity to enable development of new classes of pathway-targeted therapeutics. It may be possible in future for SCNA-modified pathways to serve as targets the same way that

**7. Potential for new treatments by targeting copy number alterations**

tics can be effective and that some could enter the clinic in the near future.

SNVs have a proven track record of targetability using small molecules. Nonetheless, in the case of SOC, new cures are unlikely to be found unless somatic copy number alterations (SCNAs) are considered. Defining this interplay will be a difficult task. It remains unclear exactly which SCNAs are most critical to SOC proliferation and metastasis. The creation of cell line models will require new methods of whole-chromosome manipulation, even as attracting pharmaceutical company support will be harder due to limited experience which such targeting strategies, as well as conservative business approaches towards eventual clinical adaptation. Nonetheless, there are reasons to be optimistic that SCNA-targeted therapeu-

Consider the abundance of SCNAs in advanced SOC relative to other cancer. The successful tumors have undergone selection. The phenotypes produced include well-known hallmarks of cancer: including cell cycle defects, heightened glucose uptake [106], spontaneous proliferative immortality [107], and dysregulated autophagy [108]. The same studies identify aneuploidyassociated characteristics which present vulnerabilities particular to these unstable cells. Perhaps the most promising vulnerability is an increased reliance on protein quality control processes such as ribosome biogenesis and maintenance factors and the cellular recycling process, autophagy. Aneuploid cells require these systems to function, and may result in a general

with invasion of adipocyte-rich regions of the omentum.

of ovarian cancer.

**6.6. Peroxisome**

SNVs do, now.

#### **6.3. p53 Interactome**

In addition to *TP53* gene mutation, serous ovarian cancer exhibits a number of p53-interacting components that are also suppressed by deletions. Among the top hits by HAPTRIG [8] include *CHEK2*, *BAX*, and *GADD45A/B* gene deletions, along with *CCNE1* and *ATR* amplification. Chk2 is a kinase which coordinates DNA repair and cell cycle arrest, in part by stabilizing p53. Bax is a pro-apoptotic Bcl-2 family member which associates with p53 to induce apoptosis [90]. The Gadd45 proteins mediate DNA damage signaling to p53 and act as tumor suppressors by leading to damage-induced senescence [91]. Conversely, an upregulated ATR network allows for potential enhancement of DNA repair pathways which lead to aneuploidy and may also lead to centrosome duplications [92]. This is further supported by a common suppression of Rad51 networks in SOC.

#### **6.4. Metabolism**

Metabolism is fundamentally disrupted in serous ovarian cancer. This may be predicted by the observation that patients with metabolic disruptions are at risk for disease, or have a predisposition to tumors to undergo metastatic growth to adipose tissue [93, 94]. A shift to glycolysis, the Warburg effect, is a general hallmark of cancer. Glycolytic shift is considered essential to provide the many constituent molecules required for cell division: nucleotides, lipids, and amino acids, moreso than simply ATP which is produced in higher quantities by oxidative phosphorylation [95]. A metabolic pathway found to be suppressed with almost equal magnitude to autophagy was the arginine and proline metabolism pathway, particularly through deletions in *SAT1* and *SAT2* and guanidinoacetate N-methyltransferase. Such deletions are predicted to reduce spermidine metabolism and polyamine formation, which is normally upregulated in tumors [96]. The reason for their ubiquitous suppression may lie in the increase in glutamate which would come from an inhibition of arginine biosynthesis, which can then be used in the TCA cycle [97].

#### **6.5. Adipocytokine**

Adipocytokine signaling and *fatty acid* metabolism was also altered, led by suppressed networks with the *CPT1B* gene and *ADH4,6,7*, and *1A*. Again, this result is unique and unexpected: *CPT1* isoforms are often upregulated in prostate cancer [98] as are ADH enzymes [99]. Dysregulation of ADH isoforms may enable acetaldehyde formation, which is oncogenic, or favor class I alcohol dehydrogenases, which are upregulated in cancerous ovarian tissue [100]. Conversely, one of the most upregulated metabolic pathways in serous ovarian cancer is glycerolipid metabolism. Upregulation is led by amplification of the *DGAT1* gene, encoding diglyceride acyltransferase, the committing step for synthesis of triglycerides and an essential reaction for the formation of adipose tissue. The pathway is further reinforced by overexpression of *LPIN1* and *LPIN3* genes. While targeting metabolism has not historically been successful in cancer treatment, overexpression of these genes may act as early identifiers of ovarian cancer.

#### **6.6. Peroxisome**

deletions, but that the pathway is redirected to allow for cell cycle progression proteins to persist and push the cell through division. The latter finding is perhaps to be expected, given that this has been established as a mechanism for tumor formation in many reviews [86–88]. Yet the proteasome may have a similar function to autophagy in suppressing aneuploidy. In a screen for mutations which are enabling for cell cycle progression in aneuploidy cells,

In addition to *TP53* gene mutation, serous ovarian cancer exhibits a number of p53-interacting components that are also suppressed by deletions. Among the top hits by HAPTRIG [8] include *CHEK2*, *BAX*, and *GADD45A/B* gene deletions, along with *CCNE1* and *ATR* amplification. Chk2 is a kinase which coordinates DNA repair and cell cycle arrest, in part by stabilizing p53. Bax is a pro-apoptotic Bcl-2 family member which associates with p53 to induce apoptosis [90]. The Gadd45 proteins mediate DNA damage signaling to p53 and act as tumor suppressors by leading to damage-induced senescence [91]. Conversely, an upregulated ATR network allows for potential enhancement of DNA repair pathways which lead to aneuploidy and may also lead to centrosome duplications [92]. This is further supported by a common

Metabolism is fundamentally disrupted in serous ovarian cancer. This may be predicted by the observation that patients with metabolic disruptions are at risk for disease, or have a predisposition to tumors to undergo metastatic growth to adipose tissue [93, 94]. A shift to glycolysis, the Warburg effect, is a general hallmark of cancer. Glycolytic shift is considered essential to provide the many constituent molecules required for cell division: nucleotides, lipids, and amino acids, moreso than simply ATP which is produced in higher quantities by oxidative phosphorylation [95]. A metabolic pathway found to be suppressed with almost equal magnitude to autophagy was the arginine and proline metabolism pathway, particularly through deletions in *SAT1* and *SAT2* and guanidinoacetate N-methyltransferase. Such deletions are predicted to reduce spermidine metabolism and polyamine formation, which is normally upregulated in tumors [96]. The reason for their ubiquitous suppression may lie in the increase in glutamate which would come from an inhibition of arginine biosynthesis,

Adipocytokine signaling and *fatty acid* metabolism was also altered, led by suppressed networks with the *CPT1B* gene and *ADH4,6,7*, and *1A*. Again, this result is unique and unexpected: *CPT1* isoforms are often upregulated in prostate cancer [98] as are ADH enzymes [99]. Dysregulation of ADH isoforms may enable acetaldehyde formation, which is oncogenic, or favor class I alcohol dehydrogenases, which are upregulated in cancerous ovarian tissue [100]. Conversely, one of the most upregulated metabolic pathways in serous ovarian cancer is glycerolipid metabolism. Upregulation is led by amplification of the *DGAT1* gene, encoding diglyceride acyltransferase, the committing step for synthesis of triglycerides and

ubiquitin-proteasomal degradation components were a top hit [89].

**6.3. p53 Interactome**

122 Ovarian Cancer - From Pathogenesis to Treatment

**6.4. Metabolism**

**6.5. Adipocytokine**

suppression of Rad51 networks in SOC.

which can then be used in the TCA cycle [97].

An unusually altered pathway in serous ovarian cancer bridges metabolism, fatty acid oxidation and proteostasis disruption: *peroxisome* biogenesis. Peroxisomes are subcellular organelles whose primary function is to metabolize reactive-oxygen species and provide lipids to other organelles [101]. This pathway is amplified in serous ovarian cancer, and lung adenocarcinoma only [8]. *PEX5*, *PEX5L*, and *PEX19* are all commonly amplified. Pex5 and Pex19 bind to peroxisome enzymes in the cytosol and direct them to the peroxisome matrix [102, 103]. In fact, amplification of PEX5 is associated with poorer outcome in SOC. Peroxiredoxin-1 is also strongly amplified, and can be detected at increased levels in ovarian cancer patients' serum [104], and is also associated with lung cancer malignancy [105]. Upregulation of this pathway, and those associated with phospholipid metabolism may provide to a means to overcome oxidative stress, perform fatty acid beta-oxidation, and resist lipotoxicity associated with invasion of adipocyte-rich regions of the omentum.

While each of these pathways can help to define phenotypes associated with SOC, they also have the capacity to enable development of new classes of pathway-targeted therapeutics. It may be possible in future for SCNA-modified pathways to serve as targets the same way that SNVs do, now.
