Prostate cancer (PCa) is one of the leading causes of cancer deaths in men. In this cancer, the stem cell transcription factor SOX2 increases during tumor progression, especially as the cancer progresses to the highly aggressive neuroendocrine-like phenotype. Other studies have shown that knockdown of RB1 and TP53 increases the expression of neuroendocrine markers, decreases the sensitivity to enzalutamide, and increases the expression of SOX2. Importantly, knockdown of SOX2 in the context of RB1 and TP53 depletion restored sensitivity to enzalutamide and reduced the expression of neuroendocrine markers. In this study, we examined whether elevating SOX2 is not only necessary, but also sufficient on its own to promote the expression of neuroendocrine markers and confer enzalutamide resistance. For this purpose, we engineered LNCaP cells for inducible overexpression of SOX2 (i-SOX2-LNCaP). As shown previously for other tumor cell types, inducible elevation of SOX2 in i-SOX2-LNCaP inhibited cell proliferation. SOX2 elevation also increased the expression of several neuroendocrine markers, including several neuropeptides and synaptophysin. However, SOX2 elevation did not decrease the sensitivity of i-SOX2-LNCaP cells to enzalutamide, which indicates that elevating SOX2 on its own is not sufficient to confer enzalutamide resistance. Furthermore, knocking down SOX2 in C4-2B cells, a derivative of LNCaP cells which is far less sensitive to enzalutamide and which expresses much higher levels of SOX2 than LNCaP cells, did not alter the growth response to this antiandrogen. Thus, our studies indicate that NE marker expression can increase independently of the sensitivity to enzalutamide.

The stem cell transcription factor Sox2 is widely recognized for its many roles during normal development and cancer. Over the last several years, it has become increasingly evident that Sox2 dosage plays critical roles in both normal and malignant cells. The work described in this review indicates that the dosage of Sox2 influences cell fate decisions made during normal mammalian development, as well as cell fate decisions in cancer, including those that influence the tumor cell of origin and progression of the cancer. Equally important, Sox2 dosage is a key determinant in the proliferation of both normal cells and tumor cells, where proliferation is restricted in Sox2 high cells. Collectively, the studies reviewed here indicate that tumor cells utilize the fundamental effects of Sox2 dosage to suit their own needs. Finally, we speculate that elevated expression of Sox2 helps establish and maintain tumor dormancy in Sox2-positive cancers. K E Y W O R D S cancer, development, dormancy, quiescence, Sox2

Background Quiescent tumor cells pose a major clinical challenge due to their ability to resist conventional chemotherapies and to drive tumor recurrence. Understanding the molecular mechanisms that promote quiescence of tumor cells could help identify therapies to eliminate these cells. Significantly, recent studies have determined that the function of SOX2 in cancer cells is highly dose dependent. Specifically, SOX2 levels in tumor cells are optimized to promote tumor growth: knocking down or elevating SOX2 inhibits proliferation. Furthermore, recent studies have shown that quiescent tumor cells express higher levels of SOX2 compared to adjacent proliferating cells. Currently, the mechanisms through which elevated levels of SOX2 restrict tumor cell proliferation have not been characterized. Methods To understand how elevated levels of SOX2 restrict the proliferation of tumor cells, we engineered diverse types of tumor cells for inducible overexpression of SOX2. Using these cells, we examined the effects of elevating SOX2 on their proliferation, both in vitro and in vivo. In addition, we examined how elevating SOX2 influences their expression of cyclins, cyclin-dependent kinases (CDKs), and p27Kip1. Results Elevating SOX2 in diverse tumor cell types led to growth inhibition in vitro. Significantly, elevating SOX2 in vivo in pancreatic ductal adenocarcinoma, medulloblastoma, and prostate cancer cells induced a reversible state of tumor growth arrest. In all three tumor types, elevation of SOX2 in vivo quickly halted tumor growth. Remarkably, tumor growth resumed rapidly when SOX2 returned to endogenous levels. We also determined that elevation of SOX2 in six tumor cell lines decreased the levels of cyclins and CDKs that control each phase of the cell cycle, while upregulating p27Kip1. Conclusions Our findings indicate that elevating SOX2 above endogenous levels in a diverse set of tumor cell types leads to growth inhibition both in vitro and in vivo. Moreover, our findings indicate that SOX2 can function as a master regulator by controlling the expression of a broad spectrum of cell cycle machinery. Importantly, our SOX2-inducible tumor studies provide a novel model system for investigating the molecular mechanisms by which elevated levels of SOX2 restrict cell proliferation and tumor growth.

Slowly cycling/infrequently proliferating tumor cells present a clinical challenge due to their ability to evade treatment. Previous studies established that high levels of SOX2 in both fetal and tumor cells restrict cell proliferation and induce a slowly cycling state. However, the mechanisms through which elevated SOX2 levels inhibit tumor cell proliferation have not been identified. To identify common mechanisms through which SOX2 elevation restricts tumor cell proliferation, we initially performed RNA-seq using two diverse tumor cell types. SOX2 elevation in both cell types downregulated MYC target genes. Consistent with these findings, elevating SOX2 in five cell lines representing three different human cancer types decreased MYC expression. Importantly, the expression of a dominant-negative MYC variant, omomyc, recapitulated many of the effects of SOX2 on proliferation, cell cycle, gene expression, and biosynthetic activity. We also demonstrated that rescuing MYC activity in the context of elevated SOX2 induces cell death, indicating that the downregulation of MYC is a critical mechanistic step necessary to maintain survival in the slowly cycling state induced by elevated SOX2. Altogether, our findings uncover a novel SOX2:MYC signaling axis and provide important insights into the molecular mechanisms through which SOX2 elevation induces a slowly cycling proliferative state.

The poly d(A-T) dependent synthesis of the analog alternating copolymer poly d(A-4thioT) (in which thymidine is replaced by 4-thiothymidine) by DNA polymerase of Escherichia coli is accompanied by hydrolysis of a t least one mole of primer d(A-T) per mole of product d(A-4thioT) formed. Exonuclease 11, the deoxyribonuclease associated with DNA polymerase, is responsible for the hydrolysis of the primer. Since the growing 3' end of the primer is protected from hydrolysis during the enzymatic polymerisation the hydrolysis must occur at the free 5' end. The enzymatic synthesis of poly d(A-4thioT) on a poly d(A-T) template proceeds in a two phase time course. The ratio of polymerisation versus hydrolysis is close to unity for the first rapid phase of synthesis. The temperature-dependence of poly d(A-T) and poly d(A-4thioT) synthesis, represented by usual Arrhenius plots has a discontinuity at 15" and 25", respectively.ExonucIease I1 exhibits a similar discontinuity a t 26" under the same conditions. Above 25", the synthesis of-poly d(A-4thioT) has a smaller apparent activation energy than the poly d(A-T) synthesis, but the latter is twice to three times as fast. The obligatory hydrolysis of the primer offers a plausible explanation of why the synthesis of poly d(A-4thioT) depends on the amount of poly d(A-T) primer in a stoichiometric manner since the former copolymer is not a primer for extensive synthesis of poly d(A4thioT). A modified enzyme model for DNA polymerase is suggested, implicating the function of exonuclease I1 in providing an additional driving force for the unidirectional movement of the enzyme along the template. Emphasis is put on the energy contribution arising from formation of new base pairs during synthesis.The specificity of exonuclease I1 was also studied using as substrates such polymers which consist of poly d(A-T) and poly d(A-4thioT) in one half each. The polymer bearing the d(A-4thioT), a t the 3' half is readily hydrolysed by exonuclease I1 from both the 3' and 5' end synchronously at pH 7.2. Upon mercuration of d(A-4thioT), with p-chloromercuribenzoate, preferentially the poly d(A-T) part is hydrolysed from the 5' end at a reduced rate. I n contrast, no synchronism of nuclease reaction is observed with the polymer bearing d(A-4thioT), at the 6' end, the latter being generally more resistant against hydrolytic attack by the enzyme. Enzymes. DNA polymerase or deoxynucleosidetriphosphate: DNA deoxynucleotidyl-transferase (EC 2.7.7.7) ; thymidine kinase (EC 2.7.1.21); thymidine monophosphate kinase (EC 2.7.4.9).feature of a nucleotide analog not present in DNA from natural sources could not be explained and subsequent studies were pursued with the aim of obtaining more insight into the enzymatic mechanism of a template-directed polymerisation in this model system. Increasing evidence for an important role of deoxyribonucleases in the process of DNA replication, as reviewed by Lehmann f2], and the surprising discovery of Klett et at.[3] that exonuclease I1 can hydrolyse poly d(A-T) from the ...