The high attrition rates of drug development projects for targeted therapies 
, necessitates identification and validation of new druggable molecular targets, with their role in ovarian cancer clearly defined to minimize failure of the drug during the development pipeline. Using an integrated RNAi screening approach to target over 6,000 druggable genes we identified 53 that were required for growth and survival across a panel of EOC cell lines; seven of these were predominantly active in tumorigenic cells and were considered for additional deconvolution and validation studies. Four candidates out of the seven (HSPA5
, and PTN
) ultimately proved to be valid hits for EOC cells with minimal effects on the non-tumorigenic HIO cells.
The loss-of-function screening studies reported in this paper have provided us with a functional genomic snapshot of novel molecular vulnerabilities in epithelial ovarian cancer outside the realm of commonly targeted molecular signaling pathways. We have studied the four validated targets (HSPA5
), all with a role in growth or survival of EOC cells, using in vitro
cell-based assays. The results show that ovarian tumorigenic cells, on average, are comparatively more vulnerable to the candidate targets compared with non-tumorigenic cells suggesting a possible therapeutic window of sensitization. All four genes have been previously reported as hits in RNA interference screens. 
All four targets code for proteins amenable to therapeutic intervention and have been previously reported to participate in cell cycle pathways or survival pathways in other tumor types. 
Genomics data from the TCGA and the Birrer lab further support the notion that for at least three of the targets (NDC80
) there should be selective vulnerability to therapeutics in tumor cells relative to normal cells given the significant up-regulation in serous adenocarcinomas. 
Currently, the most promising inhibitors targeting these candidates include INH11 
which targets the NDC80/NUF2 pathway, the neutralizing anti-PTN antibodies 
which functionally inhibit the tumor growth promoting activities of PTN, and epigallocatechin gallate which inhibits HSPA5. 
Additionally, siRNA-based drugs have also proven to be feasible options for in vivo
providing us with avenues to proceed with preclinical studies to measure the effectiveness of targeting our four hits using orthotopic, xenograft mouse models of ovarian cancer.
() is a gene whose product is a central regulator for endoplasmic reticulum homeostasis which is critical for the survival of eukaryotic cells. 
HSPA5 is a stress-inducible ER chaperone that is highly induced in a wide range of tumors through factors like hypoxia and acidosis in the microenvironment of poorly perfused tumors. 
In a previous study, antibodies targeting cell surface HSPA5 induced apoptosis in SKOV3 cells. 
In the current study, silencing of HSPA5 induced significant apoptosis in A2780 cells and showed a significant cell cycle arrest of A1847 cells in the G1 phase. Gene expression data from TCGA suggest that reduced expression is more common for this gene, which is counter to our screening results. CNV analysis shows that HSPA5
is lost in 41% of the samples and mutational analysis of TCGA data shows that HSPA5
is mutated in less than 1% of samples. Given the reduced expression, gene deletion, and lack of mutations in the tumor samples from ovarian cancer patients, additional studies are required in order to gain a better understanding of the mechanism of action and the clinical significance of this hit for ovarian cancer.
Consistent with our screening data, NDC80
are overexpressed in nearly 100% of the samples and PTN
is overexpressed in ~40% of the samples for two independent cohorts of patient samples. The protein products of NDC80
) and NUF2
) are part of a mitotic complex involved in kinetochore interactions and the spindle assembly checkpoint in mitosis. 
Mitosis dysregulation is a common cause in carcinogenesis. 
In a previous study, siRNA mediated knockdown against NDC80
has been shown to cause abnormal mitotic exit and induce apoptosis in colorectal cancer and gastric cancer cell lines. 
In another study silencing of NDC80
in an EOC cell line, SKOV3.ip1, suggested that an increase in apoptosis-related cell death. 
have been shown to be up regulated in brain, liver, and breast cancer. 
Over-expression of NDC80
has been related to poor clinical prognosis in patients with breast cancers and non-small cell lung cancers 
. Disruption of NDC80 and NUF2 complex formation using a small molecule inhibitor, INH1, has been shown to reduce proliferation in breast cancer cells and reduce tumor growth in a xenograft mouse model. 
Kinetochore components, particularly NDC80 and NUF2, have been proposed as potential targets for cancer therapeutics. 
Our study represents the first report on NDC80 and NUF2 as potential drug targets for treatment of ovarian cancer.
) is another interesting gene identified whose product is a growth factor known to elicit downstream survival signaling pathways through multiple receptors namely ALK, SDC3, SDC1 and PTPRb/z. 
It has been shown to play a pivotal role in tumorigenesis in pancreatic, brain and breast tumor models. 
It is involved in cell transformation, growth, survival, migration and angiogenesis. The PTN
gene is highly expressed during embryogenesis but shows very limited expression in adult tissues, where it is restricted to the brain. 
We have shown using ELISA assays that PTN levels are significantly elevated in conditioned media of the ovarian cancer cell lines examined (G. Sethi and A.K. Godwin, unpublished data). This makes it an attractive therapeutic target for ovarian cancer as anti-PTN therapeutics are expected to show high efficacy with minimal side effects on non-tumorigenic cells. Our study is the first to show that PTN
is required for growth and survival of ovarian tumor cells.
It is now well established that both oncogenic and non-oncogenic addictions contribute to the extensively rewired pathways that underlie the malignant phenotype in cancer cells. 
We have concentrated on genes which have activities across multiple ovarian cancer cell lines representing primarily the serous subtype. Future studies which expand our screening panel to include additional cell lines which represent other EOC subtypes (clear cell, endometrioid, and mucinous) should provide us with subtype related/specific sensitization patterns that can further be explored. In addition, we will need to establish if any or all of the validated targets have oncogenic properties, the efficacy of targeting these candidates in vivo
, and whether targeting these candidates exhibits “genotype dependent lethality” 
that exploits the enhanced sensitivity of cancer cells to DNA damage. As we continue to move towards better treatments for ovarian cancer patients, it will be essential to clearly define critical and functional nodes whose perturbation will lead to cancer cell lethality.