High-throughput ectopic expression screen for tamoxifen resistance identifies an atypical kinase that blocks autophagy.

Resistance to tamoxifen in breast cancer patients is a serious therapeutic problem and major efforts are underway to understand underlying mechanisms. Resistance can be either intrinsic or acquired. We derived a series of subcloned MCF7 cell lines that were either highly sensitive or naturally resistant to tamoxifen and studied the factors that lead to drug resistance. Gene-expression studies revealed a signature of 67 genes that differentially respond to tamoxifen in sensitive vs. resistant subclones, which also predicts disease-free survival in tamoxifen-treated patients. High-throughput cell-based screens, in which >500 human kinases were independently ectopically expressed, identified 31 kinases that conferred drug resistance on sensitive cells. One of these, HSPB8, was also in the expression signature and, by itself, predicted poor clinical outcome in one cohort of patients. Further studies revealed that HSPB8 protected MCF7 cells from tamoxifen and blocked autophagy. Moreover, silencing HSBP8 induced autophagy and caused cell death. Tamoxifen itself induced autophagy in sensitive cells but not in resistant ones, and tamoxifen-resistant cells were sensitive to the induction of autophagy by other drugs. These results may point to an important role for autophagy in the sensitivity to tamoxifen.

Kinase requirements in human cells: V. Synthetic lethal interactions between p53 and the protein kinases SGK2 and PAK3.

Cervical carcinomas are initiated through a series of well-defined stages that rely on the expression of human papillomavirus (HPV) oncogenes. A panel of 100 small hairpin RNAs that target essential kinases in many tumor types was used to study the stepwise appearance of kinase requirements during cervical tumor development. Twenty-six kinases were commonly required in three cell lines derived from frank carcinomas, and each kinase requirement was traced to the specific stage in which the requirement emerged. Six kinases became required following HPV-induced immortalization, and the requirement for two kinases, SGK2 and PAK3, was mapped to the inactivation of p53 in primary human epithelial cells. Loss of the p53 tumor suppressor in other primary epithelial cells also induced dependence on SGK2 and PAK3. Hence, SGK2 and PAK3 provide important cellular functions following p53 inactivation, fulfilling the classical definition of synthetic lethality; loss of p53, SGK2, or PAK3 alone has little effect on cell viability, whereas loss of p53 together with either SGK2 or PAK3 loss leads to cell death. Whereas tumor suppressor gene mutations are not directly druggable, other proteins or pathways that become obligatory to cell viability following tumor suppressor loss provide theoretical targets for tumor suppressor-specific drug discovery efforts. The kinases SGK2 and PAK3 may thus represent such targets for p53-specific drug development.

Kinase requirements in human cells: IV. Differential kinase requirements in cervical and renal human tumor cell lines.

Functional differences among human cells have been difficult to identify by standard biochemical methods. Loss-of-function shRNA screens provide an unbiased method to compare protein requirements across cell lines. In previous work, we have studied kinase requirements in two settings, either among a panel of cells from numerous tissues or between two cell lines that differ only by the expression of a chosen oncoprotein or tumor suppressor protein. Here we examine the patterns of kinase requirements between two unrelated cells, the cervical carcinoma cell line HeLa and the renal carcinoma cell line 786-O. By using time courses of cell proliferation after shRNA transduction and by introducing different levels of the shRNAs, we were able to carefully compare the kinase requirements. These comparisons identified 10 kinases that were required in HeLa but not 786-O, and 5 kinases that were required in 786-O but not HeLa. The patterns of growth inhibition due to particular sets of shRNAs in a tumor cell line were shown to be similar in some but not all cell lines derived from the same tissue-specific cancer type. Differential kinase requirements promise to be useful in distinguishing important cell-to-cell functional variations and may lead to the identification of fingerprints for different physiological cell states.

Kinase requirements in human cells: III. Altered kinase requirements in VHL-/- cancer cells detected in a pilot synthetic lethal screen.

Clear cell renal carcinomas are the most common form of kidney cancer and frequently are linked to biallelic inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene product, pVHL, has multiple functions including directing the polyubiquitylation of the HIF transcription factor. We screened 100 shRNA vectors, directed against 88 kinases, for their ability to inhibit the viability of VHL-/- renal carcinoma cells preferentially compared with isogenic cells in which pVHL function was restored. shRNAs for "hits" identified in the primary screen were interrogated in secondary screens that included shRNA titration studies. Multiple shRNAs against CDK6, MET, and MAP2K1 (also known as MEK1) preferentially inhibited the viability of 786-O and RCC4 VHL-/- cells compared with their wild-type pVHL-reconstituted counterparts. The sensitivity of pVHL-proficient cells to these shRNAs was not restored upon HIF activation, suggesting that loss of an hypoxia-inducible factor (HIF)-independent pVHL function formed the basis for selectivity. A small-molecule Cdk4/6 inhibitor displayed enhanced activity against VHL-/- renal carcinoma cells, suggesting that in some cases hits from shRNA screens such as described here might translate into therapeutic targets.

Kinase requirements in human cells: I. Comparing kinase requirements across various cell types.

shRNA loss-of-function screens were used to identify kinases that were rate-limiting for promoting cell proliferation and survival. Here, we study the differences in kinase requirements among various human cells, including freshly prepared primary cells, isogenic cells, immortalized cells, and cancer cell lines. Closely related patterns of kinase requirements among the various cell types were observed in three cases: (i) in repeat experiments using the same cells, (ii) with multiple populations of freshly prepared primary epithelial cells isolated from the same tissue source, and (iii) between nearly isogenic cells that differ from each other by the expression of a single gene. Other commonly used cancer cell lines were distinct from one another, even when they were isolated from similar tumor types. Even primary cells of different lineages isolated from the same tissue source showed many differences. The differences in kinase requirements among cell lines observed in this study suggest that the control of proliferation and survival may be significantly different between cell lines and that simple comparisons from any one cell to another may be misleading. Although the regulation of cell proliferation and survival are heavily studied areas, we did not see a bias in these screens toward the identification of previously known and well studied kinases, suggesting that our knowledge of molecular events in these areas is still meager.